PSCA: prostate stem cell antigen and uses thereof

ABSTRACT

The invention provides a novel prostate cell-surface antigen, designated Prostate Stem Cell Antigen (PSCA), which is widely over-expressed across all stages of prostate cancer, including high grade prostatic intraepithelial neoplasia (PIN), androgen-dependent and androgen-independent prostate tumors.

[0001] This application is a continuation-in-part (CIP) of U.S. Ser. No.09/359,326, filed Jul. 20, 1999, which is a CIP of U.S. Ser. No.09/308,503, filed May 25, 1999, which is a CIP of U.S. Ser. No.09/251,835, filed Feb. 17, 1999, which is a continuation-in-part (CIP)of U.S. Ser. No. 09/203,939, filed Dec. 2, 1998, which is a CIP of U.S.Ser. No. 09/038,261, filed Mar. 10, 1998; claiming the priority ofprovisional applications, U.S. Ser. No. 08/814,279, filed Mar. 10, 1997;U.S. Ser. No. 60/071,141 filed Jan. 12, 1998 and; U.S. Ser. No.60/074,675, filed Feb. 13, 1998. This application further claims thebenefit of the filing dates of U.S. Serial Nos. 60/124,658 filed Mar.16, 1999; 60/120,536 filed Feb. 17, 1999; and 60/113,230 filed Dec. 21,1998. The contents of all of the foregoing applications are incorporatedby reference into the present application.

[0002] Throughout this application, various publications are referencedwithin parentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of human death next tocoronary disease. Worldwide, millions of people die from cancer everyyear. In the United States alone, cancer cause the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

[0004] Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the leading causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients that initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience significant physical debilitations followingtreatment.

[0005] Generally speaking, the fundamental problem in the management ofthe deadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

[0006] Recently, there has been a particularly strong interest inidentifying cell surface tumor-specific antigens which might be usefulas targets for various immunotherapeutic or small molecule treatmentstrategies. A large number of such cell-surface antigens have beenreported, and some have proven to be reliably associated with one ormore cancers. Much attention has been focused on the development ofnovel therapeutic strategies which target these antigens. However, fewtruly effective immunological cancer treatments have resulted.

[0007] The use of monoclonal antibodies to tumor-specific orover-expressed antigens in the treatment of solid cancers isinstructive. Although antibody therapy has been well researched for some20 years, only very recently have corresponding pharmaceuticalsmaterialized. One example is the humanized anti-HER2/neu monoclonalantibody, Herceptin, recently approved for use in the treatment ofmetastatic breast cancers overexpressing the HER2/neu receptor. Anotheris the human/mouse chimeric anti-CD20/B cell lymphoma antibody, Rituxan,approved for the treatment of non-Hodgkin's lymphoma. Several otherantibodies are being evaluated for the treatment of cancer in clinicaltrials or in pre-clinical research, including a chimeric and a fullyhuman IgG2 monoclonal antibody specific for the epidermal growth factorreceptor (Slovin et al., 1997, Proc. Am. Soc. Clin. Oncol. 16:311;Falcey et al., 1997, Proc. Am. Soc. Clin. Oncol. 16:383; Yang et al.,1999, Cancer Res. 59: 1236). Evidently, antibody therapy is finallyemerging from a long embryonic phase. Nevertheless, there is still avery great need for new, more-specific tumor antigens for theapplication of antibody and other biological therapies. In addition,there is a corresponding need for tumor antigens which may be useful asmarkers for antibody-based diagnostic and imaging methods, hopefullyleading to the development of earlier diagnosis and greater prognosticprecision.

[0008] As discussed below, the management of prostate cancer serves as agood example of the limited extent to which molecular biology hastranslated into real progress in the clinic. With limited exceptions,the situation is more or less the same for the other major carcinomasmentioned above.

[0009] Worldwide, prostate cancer is the fourth most prevalent cancer inmen. In North America and Northern Europe, it is by far the most commonmale cancer and is the second leading cause of cancer death in men. Inthe United States alone, well over 40,000 men die annually of thisdisease, second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy remain as the main treatment modalities.Unfortunately, these treatments are clearly ineffective for many.Moreover, these treatments are often associated with significantundesirable consequences.

[0010] On the diagnostic front, the serum PSA assay has been a veryuseful tool. Nevertheless, the specificity and general utility of PSA iswidely regarded as lacking in several respects. Neither PSA testing, norany other test nor biological marker has been proven capable of reliablyidentifying early-stage disease. Similarly, there is no marker availablefor predicting the emergence of the typically fatal metastatic stage ofthe disease. Diagnosis of metastatic prostate cancer is achieved by opensurgical or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy analysis.Clearly, better imaging and other less invasive diagnostic methods offerthe promise of easing the difficulty those procedures place on apatient, as well as improving therapeutic options. However, until thereare prostate tumor markers capable of reliably identifying early-stagedisease, predicting susceptibility to metastasis, and precisely imagingtumors, the management of prostate cancer will continue to be extremelydifficult. Accordingly, more specific molecular tumor markers areclearly needed in the management of prostate cancer.

[0011] There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54:1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252). PCTA-1, anovel galectin, is largely secreted into the media of expressing cellsand may hold promise as a diagnostic serum marker for prostate cancer(Su et al., 1996). Vaccines for prostate cancer are also being activelyexplored with a variety of antigens, including PSM and PSA.

SUMMARY OF THE INVENTION

[0012] The invention provides a novel prostate cell-surface antigen,designated Prostate Stein Cell Antigen (PSCA), which is widelyover-expressed across all stages of prostate cancer, including highgrade prostatic intraepithelial neoplasia (PIN), androgen-dependent andandrogen-independent prostate tumors. The PSCA gene shows 30% homologyto stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly-6 family ofglycosylphosphatidylinositol (GPI)-anchored cell surface antigens, andencodes a 123 amino acid protein with an amino-terminal signal sequence,a carboxy-terminal GPI-anchoring sequence, and multiple N-glycosylationsites. PSCA mRNA expression is highly upregulated in both androgendependent and androgen independent prostate cancer xenografts. In situmRNA analysis localizes PSCA expression to the basal cell epithelium,the putative stem cell compartment of the prostate. Flow cytometricanalysis demonstrates that PSCA is expressed predominantly on the cellsurface and is anchored by a GPI linkage. Fluorescent in situhybridization analysis localizes the PSCA gene to chromosome 8q24.2, aregion of allelic gain in more than 80% of prostate cancers.

[0013] PSCA may be an optimal therapeutic target in view of its cellsurface location, greatly upregulated expression in certain types ofcancer such as prostate cancer cells. In this regard, the inventionprovides antibodies capable of binding to PSCA which can be usedtherapeutically to destroy or inhibit the growth of such cancer cells,or to block PSCA activity. In addition, PSCA proteins and PSCA-encodingnucleic acid molecules may be used in various immunotherapeutic methodsto promote immune-mediated destruction or growth inhibition of tumorsexpressing PSCA.

[0014] PSCA also may represent an ideal prostate cancer marker, whichcan be used to discriminate between malignant prostate cancers, normalprostate glands and non-malignant neoplasias. For example, PSCA isexpressed at very high levels in prostate cancer in relation to benignprostatic hyperplasia (BPH). In contrast, the widely used prostatecancer marker PSA is expressed at high levels in both normal prostateand BPH, but at lower levels in prostate cancer, rendering PSAexpression useless for distinguishing malignant prostate cancer from BPHor normal glands. Because PSCA expression is essentially the reverse ofPSA expression, analysis of PSCA expression can be employed todistinguish prostate cancer from non-malignant conditions.

[0015] The genes encoding both human and murine PSCA have been isolatedand their coding sequences elucidated and provided herein. Also providedare the amino acid sequences of both human and murine PSCA. Theinvention further provides various diagnostic assays for the detection,monitoring, and prognosis of prostate cancer, including nucleicacid-based and immunological assays. PSCA-specific monoclonal andpolyclonal antibodies and immunotherapeutic and other therapeuticmethods of treating prostate cancer are also provided. These and otheraspects of the invention are further described below.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1. Nucleotide (A) and translated amino acid (B) sequences ofa cDNA encoding human PSCA (ATCC Designation 209612).

[0017]FIG. 2. Nucleotide sequence of a cDNA encoding murine PSCAhomologue.

[0018]FIG. 3. Alignment of amino acid sequences of human PSCA, murinePSCA, and human stem cell antigen-2 (hSCA-2). Shaded regions highlightconserved amino acids. Conserved cysteines are indicated by boldlettering. Four predicted N-glycosylation sites in PSCA are indicated byasterisks. The underlined amino acids at the beginning and end of theprotein represent N terminal hydrophobic signal sequences and C terminalGPI-anchoring sequences, respectively.

[0019]FIG. 4. Hydrophobicity plot of human PSCA.

[0020]FIG. 5. Chou-Fassman analysis of human PSCA.

[0021]FIG. 6. A western blot showing that monoclonal antibody 1G8 bindsLAPC9 (PSCA positive control) and a transitional cell carcinoma (bladdercarcinoma) designated bladder (Rob).

[0022]FIG. 7. Restricted Expression of PSCA mRNA in normal and canceroustissues. A: RT-PCR analysis of PSCA expression in normal human tissuesdemonstrating high expression in prostate, placenta, and tonsils. 1 ngof reverse-transcribed first strand cDNA (Clontech, Palo Alto, Calif.)from the indicated tissues was amplified with PSCA gene specificprimers. Data shown are from 30 cycles of amplification. B: RT-PCRanalysis of PSCA expression demonstrating high level in prostate cancerxenografts and normal tissue. 5 ng of reverse-transcribed cDNA from theindicated tissues were amplified with PSCA gene specific primers.Amplification with β-actin gene specific primers demonstratenormalization of the first strand cDNA of the various samples. Datashown are from 25 cycles of amplification. AD, androgen-dependent; AL,androgen-independent; IT, intratibial xenograft; C.L., cell line.

[0023]FIG. 8. Schematic representation of human PSCA, murine PSCA, andhuman Thy-1/Ly-6 gene structures.

[0024]FIG. 9. Northern blot analysis of PSCA RNA expression. A: TotalRNA from normal prostate and LAPC-4 androgen dependent (AD) andindependent (AI) prostate cancer xenografts were analyzed using PSCA orPSA specific probes. Equivalent RNA loading and RNA integrity weredemonstrated separately by ethidium staining for 18S and 28S RNA. B:Human multiple tissue Northern blot analysis of PSCA RNA. The filter wasobtained from Clontech (Palo Alto, Calif.) and contains 2 ug of polyARNA in each lane.

[0025]FIG. 10. Northern blot comparison of PSCA, PSMA, and PSA RNAexpression in prostate cancer xenografts and tumor cell lines. PSCA andPSMA demonstrate high level prostate cancer specific gene expression. 10μg of total RNA from the indicated tissues were size fractionated on anagarose/formaldehyde gel, transferred to nitrocellulose, and hybridizedsequentially with ³²P-labelled probes representing PSCA, PSMA, and PSAcDNA fragments. Shown are 4 hour and 72 hour autoradiographic exposuresof the membrane and the ethidium bromide gel demonstrating equivalentloading of samples. BPH, benign prostatic hyperplasia; AD,androgen-dependent; AI, androgen-independent; IT, intratibial xenograft;C.L., cell line.

[0026]FIG. 11. In situ hybridization with antisense riboprobe for humanPSCA RNA on normal and malignant prostate specimens. A: PSCA RNA isexpressed by a subset of basal cells within the basal cell epithelium(black arrows), but not by the terminally differentiated secretory cellslining the prostatic ducts (400×magnification). B: PSCA RNA is expressedstrongly by a high grade prostatic intraepithelial neoplasia (P1IN)(black arrow) and by invasive prostate cancer glands (yellow arrows),but is not detectable in normal epithelium (green arrow) at40×magnification. C: Strong expression of PSCA RNA in a case of highgrade carcinoma (200×magnification).

[0027]FIG. 12. Biochemical analysis of PSCA protein. A: PSCA protein wasimmunoprecipitated from 293T cells transiently transfected with a PSCAconstruct and then digested with either N-glycosidase F orO-glycosidase, as described in Materials and Methods. B: PSCA proteinwas immunoprecipitated from 293T transfected cells, as well as fromconditioned media of these cells. Cell-associated PSCA migrates higherthan secreted or shed PSCA on a 15% polyacrylamide gel. C:FACS analysisof mock-transfected 293T cells, PSCA-transfected 293T cells and LAPC-4prostate cancer xenograft cells using an affinity purified polyclonalanti-PSCA antibody. Cells were not permeabilized in order to detect onlysurface expression. The y axis represents relative cell number and the xaxis represents fluorescent staining intensity on a logarithmic scale.

[0028]FIG. 13. In situ hybridization of biotin-labeled PSCA probes tohuman metaphase cells from phytohemagglutinin-stimulated peripheralblood lymphocytes. The chromosome 8 homologues are identified witharrows; specific labeling was observed at 8q24.2. The inset showspartial karyotypes of two chromosome 8 homologues illustrating specificlabeling at 8q24.2 (arrowheads). Images were obtained using a ZeissAxiophot microscope coupled to a cooled charge coupled device (CCD)camera. Separate images of DAPI stained chromosomes and thehybridization signal were merged using image analysis software (NU200and Image 1.57).

[0029]FIG. 14. Flow Cytometric analysis of cell surface PSCA proteinexpression on prostate cancer xenograft (LAPC-9), prostate cancer cellline (LAPC-4) and normal prostate epithelial cells (PreC) usinganti-PSCA monoclonal antibodies 1G8 (green) and 3E6 (red), mouseanti-PSCA polyclonal serum (blue), or control secondary antibody(black). See Example 5 for details.

[0030]FIG. 15. (a) An epitope map for each of the seven disclosedantibodies. (b) Epitope mapping of anti-PSCA monoclonal antibodiesconducted by Western blot analysis of GST-PSCA fusion proteins.

[0031]FIG. 16. A schematic diagram showing that PSCA is a GPI-anchoredprotein.

[0032]FIG. 17. A photograph showing a FISH analysis of PSCA and c-mycGene Copy No. in prostate cancer.

[0033]FIG. 18. A photograph showing FITC labeled 1G8 antibodies stronglybind PSCA protein on PSCA transfected LNCAP cells.

[0034]FIG. 19. A photograph showing FITC labeled 1G8 antibodies weaklybind PreC cells.

[0035]FIG. 20. A photograph showing in situ RNA hybridization of PSCA innormal prostate basal cells.

[0036]FIG. 21. PSCA immunostaining in primary prostate cancers.Representative paraffin-embedded sections from four patients werestained with anti-PSCA mAbs. The specimen from patient 1 demonstratesoverexpression of PSCA protein in a Gleason grade 4 tumor (arrow) andundetectable expression of PSCA in adjacent normal glands (arrowhead)using PSCA mAb 1G8. The positively staining cancer completely surroundsthe normal glands. The specimen from patient 2 demonstratesheterogeneous staining in a Gleason grade 3+¾ cancer. The Gleasonpattern 3 glands (arrowhead) stain weakly compared with the larger, morecribriform appearing Gleason pattern ¾ glands (arrow). The specimen frompatient 3 demonstrates strong expression of PSCA by a poorlydifferentiated Gleason 5 (arrow) tumor with mAb 1G8. Patient 4 is abiopsy specimen showing no PSCA staining in the majority of a poorlydifferentiated tumor (arrowhead) and extremely weak staining in acribriform focus identified in the specimen. The matched bone metastasisfrom patient 4 is shown in FIG. 28.

[0037]FIG. 22. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 1G8 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

[0038]FIG. 23. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 1G8 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

[0039]FIG. 24. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 3E6 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

[0040]FIG. 25. A northern blot showing increased level of PSCA RNA inLAPC9 and transitional cell carcinoma of an advanced bladder carcinoma.

[0041]FIG. 26. A photograph of a tissue undergoing early stage prostatecancer as determined by biotinylated 3E6 monoclonal antibody linked tohorseradish peroxidase-conjugated streptavidin.

[0042]FIG. 27. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by hematoxylin stained 3E6 monoclonalantibody.

[0043]FIG. 28. PSCA immunostaining in prostate cancer bone metastases.The top panel shows the hematoxylin and eosin (left) and PSCA (right)staining of a bony lesion from patient 5. A single focus suspicious forcancer (arrow) was identified in the H and E section and confirmed byintense staining with anti-PSCA mAb 1G8 (arrow). The bottom panel showsthe H and E (left) and PSCA staining of a bone lesion from patient 4.The primary lesion from patient 4 is depicted in FIG. 21. Both the H andE and PSCA stains show diffuse bony involvement by prostate cancer(arrows). Again, PSCA immunostaining in the bone metastasis is uniformand intense.

[0044]FIG. 29. A photograph of a tissue undergoing early stage prostatecancer as determined by biotinylated 1G8 monoclonal antibody linked tohorseradish peroxidase-conjugated streptavidin.

[0045]FIG. 30. A photograph showing that 1G8 binds LAPC9 cells asdetermined by hematoxylin staining.

[0046]FIG. 31. A photograph showing that 1G8 binds PSCA-transfectedLnCaP cells.

[0047]FIG. 32. A photograph showing that 1G8 does not bind LnCaP cells(not transfected with PSCA).

[0048]FIG. 33. Flow cytometric recognition of PSCA on the cell surfaceof nonpermeabilized LAPC-9 human prostate cancer cells using mAbs 1G8,2H9, 3E6, 3C5 and 4A10. Staining was compared to an irrelevant isotypecontrol antibody.

[0049]FIG. 34. A photograph showing 293T cells transiently transfectedwith PSCA and immunoblotted with PSCA monoclonal antibodies. Monoclonalantibodies 2H9 and 3E6 binds deglycosylated PSCA but does not bindglycosylated PSCA in 293T cells. In contrast, monoclonal antibodies 1G8,3C5,and 4A10 recognizes glycosylated PSCA.

[0050]FIG. 35. Immunofluorescent analysis demonstrating cell surfaceexpression of PSCA in nonpermeabilized prostate cancer cells. LNCaPcells were stably transfected with PSCA and stained with mAbs 1G8, 3E6,3C5 and 4A10. Negative controls included irrelevant isotype antibody andLNCaP cells transfected with control vector, all of which showed nostaining even after prolonged exposures.

[0051]FIG. 36. A photograph showing monoclonal antibody 2H9 binds LAPC9cells.

[0052]FIG. 37. A photograph showing immunological reactivity ofanti-PSCA mAbs. (A) Immunoprecipitation of PSCA from 293T cellstransiently transfected with PSCA using mAbs 1G8, 2H9, 3C5, 3E6 and4A10. The control was an irrelevant murine IgG mAb. (B) Immunoblotanalysis of 293T cells transiently transfected with PSCA using the fiveanti-PSCA mAbs. mAbs 1G8, 3C5 and 4A10 all recognize equivalentmolecular forms of PSCA, whereas mAbs 2H9 and 3E6 only weakly recognizedeglycosylated forms of PSCA in 293T-PSCA cells in this assay.

[0053]FIG. 38. Immunohistochemical staining of normal prostate withanti-PSCA mAbs. Examples shown include a normal gland stained with anirrelevant isotype antibody as a negative control (arrow), PSCA mAb 3E6and mAb 1G8. PSCA mAb 3E6 preferentially stains basal cells (arrow) whencompared with secretory cells (arrowhead), whereas mAb 1G8 stains bothbasal (arrow) and secretory (arrowhead) cells equally. Also shown isstrong staining of an atrophic single-layered gland from a normalprostate specimen stained with PSCA mAb 2H9.

[0054]FIG. 39. Expression of PSCA protein in normal tissues. (A) Panel ashows staining of bladder transitional epithelium with mAb 1G8. Panel bshows colonic neuroendocrine cell staining with mAb 1G8. Double stainingwith chromogranin confirmed that the positive cells are ofneuroendocrine origin (not shown). Panel c shows staining of collectingducts (arrow) and tubules with mAb 3E6. Panel d show staining ofplacental trophoblasts with mAb 3E6. (B) Northern blot analysis of PSCAmRNA expression. Total RNA from normal prostate, kidney, bladder and theLAPC-9 prostate cancer xenograft was analyzed using a PSCA specificprobe (top panel). The same membrane was probed with actin to control ofloading differences (bottom panel).

[0055]FIG. 40. Targeting of mouse PSCA gene. (A) Panel a is a schematicdrawing showing a strategy for creating a PSCA targeting vector. (B)Panel b is a photograph of a southern blot analysis of genomic DNA using3′ probe showing recovery of wild-type (+/+) and heterozygous (+/−) EScells.

[0056]FIG. 41. The upper panel is a schematic drawing of a strategy forgenerating transgenic mouse models of prostate cancer. The lower panelis a list of existing transgenic mouse models of prostate cancer.

[0057]FIG. 42. A schematic drawing showing reporter gene constructs fortransfection assay.

[0058]FIG. 43. A bar graph showing the tissue-predominant expression(prostate and bladder cells) of the 9kb human PSCA upstream regulatoryregion having increased gene expression activity.

[0059]FIG. 44. Bar graphs identifying prostate-predominant expressionelements within PSCA upstream regions having increased gene expressionactivity, i.e., the 9kb, 6kb, 3kb, and 1kb PSCA regions.

[0060]FIG. 45. A schematic drawing showing the design of transgenicvectors containing either a 9 kb or 6 kb human PSCA upstream regionoperatively linked to a detectable marker.

[0061]FIG. 46. Photographs showing that the 9kb PSCA upstream regiondrives reporter gene expression in prostate, bladder and skin in vivo.

[0062]FIG. 47. Photographs of multiple tissue northern blot analysisshowing tissue specific expression patterns of human and murine PSCARNA.

[0063]FIG. 48. Complete inhibition of LAPC-9 prostate tumor growth inSCID mice by treatment with anti-PSCA monoclonal antibodies. The upperpanel represents mice injected with LAPC-9 s.c. and treated with a mouseIgG control, while in the lower panel mice were injected with LAPC-9s.c. but treated with the anti-PSCA mAb cocktail. Each data pointrepresents the ellipsoidal volume of tumors at specified time points asdescribed in Example 18-A, infra. In the anti-PSCA group, an arbitraryvalue of 20 was given for all data points to create a line, although theactual tumor volume was 0 (Example 18-A, infra).

[0064]FIG. 49. Characteristics of anti-PSCA monoclonal antibodiesutilized in the in vivo tumor challenge study described in Example 18.(A) Isotype and epitope map: The region of PSCA protein recognized bythe anti-PSCA mAbs was determined by ELISA analysis using GST-fusionproteins (50 ng/well) encoding the indicated amino acids of PSCA.Following incubation of wells with hybridoma supernatants,anti-mouse-HRP conjugate antibody was added and reactivity wasdetermined by the addition of 3,3′ 5,5′-Tetramethylbenzidine base (TMB)substrate. Optical densities (450 nm) are the means of duplicatedeterminations. (B) Epitope map determined by Western analysis: 50 ng ofthe indicated GST-PSCA fusion protein was separated by SDS-PAGE andtransferred to nitrocellulose. Western analysis was carried out byincubation of blots with hybridoma supernatants followed byanti-mouse-HRP secondary Ab and visualized by enhanced chemiluminesence.

[0065]FIG. 50. Schematic representations of PSCA Capture ELISA. (A)Standardization and control antigens: A GST-fusion protein encodingamino acids 18-98 of the PSCA protein is used for generating a standardcurve for quantification of unknown samples. Also depicted areapproximate epitope binding regions of the anti-PSCA monoclonal andpolyclonal antibodies used in the ELISA. A secreted recombinantmammalian expressed form of PSCA is used for quality control of theELISA assay. This protein contains an Ig leader sequence to directsecretion of the recombinant protein and MYC and 6XHIS epitope tags foraffinity purification. (B) ELISA format schematic.

[0066]FIG. 51. Quantification of recombinant secreted PSCA protein. (A)PSCA capture ELISA standard curve. (B) Quantification of PSCA proteinsecreted by mammalian cells. 2 day conditioned tissue culturesupernatants from either 293T cells transfected with empty vector orwith vector encoding recombinant secreted PSCA (secPSCA) was mixed withan equal volume of either PBS or normal human serum (Omega Scientific)and analyzed for the presence of PSCA protein. Data are the means ofduplicate determinations±range. ND not detectable.

[0067]FIG. 52. Immunohistochemical Analysis of cell pellet, LAPC9ADxenograft, a BPH sample, and a prostate carcinoma tissue using anti-PSCAmonoclonal antibody 3C5.

[0068]FIG. 53. Inhibition of,LAPC-9 tumor growth by anti-PSCA monoclonalantibodies. The top panel represents mice injected with 1×10⁶ LAPC-9s.c. and treated with a mouse IgG control (n=10), the middle panelrepresents mice injected with LAPC-9 s.c. and treated with anti-PSCA mAbcocktail (n=10), the bottom panel represents mice injected with LAPC-9s.c. and treated with bovine IgG (n=5). Each data point represents theellipsoidal volume of tumors at specified time points as described inExample 18-B.

[0069]FIG. 54. Inhibition of LAPC-9 tumor growth by the anti-PSCAmonoclonal antibody 1G8. The upper panel represents mice injected with1×10⁶ LAPC-9 s.c. and treated with a mouse IgG control (n=6), while inthe lower panel mice were injected with LAPC-9 s.c. but treated with theanti-PSCA mAb 1G8 (n=7). Each data point represents the ellipsoidalvolume of tumors at specified time points.

[0070]FIG. 55. Inhibition of LAPC-9 tumor growth by anti-PSCA monoclonalantibodies 2A2 and 2H9. The upper panel represents mice injected with1×10⁶ LAPC-9 s.c. and treated with either a mouse IgG control (n=6) orthe 2A2 mAb (n=7). The lower panel represents mice injected with LAPC-9s.c. and treated with the same mouse IgG control (n=6) or the 2H9 mAb(n=7). All data points represent the mean ellipsoidal volume of tumors(mm³) at the specified time points. Error bars represent standard errorof the mean (SEM).

[0071]FIG. 56. Circulating PSA levels in LAPC-9 tumor-injected miceafter treatment with anti-PSCA mAbs 2A2 and 2H9. The upper panelrepresents the mice injected with 1×10⁶ LAPC-9 s.c. and treated witheither the mouse IgG control (n=6) or the 2A2 mAb (n =7). The lowerpanel represents mice injected with LAPC-9 s.c. but treated with eitherthe same mouse IgG control (n=6) or the 2H9 mAb (n=7). Each data pointrepresents the mean PSA level determined from the serum of mice atweekly time points. Error bars represent standard error of the mean(SEM).

[0072]FIG. 57. Inhibition of established LAPC-9 prostate cancerxenografts by PSCA monoclonal antibody 3C5. See Example 18-C4 fordetails.

[0073]FIG. 58. Amino acid sequence of the heavy chain variable domainregions of PSCA monoclonal antibodies 1G8. CDRs are labeled andunderlined.

[0074]FIG. 59. Amino acid sequence of the heavy chain variable domainregions of PSCA monoclonal antibodies 4A10. CDRs are labeled andunderlined.

[0075]FIG. 60. Amino acid sequence of the heavy chain variable domainregions of PSCA monoclonal antibodies 2H9. CDRs are labeled andunderlined.

[0076]FIG. 61. Amino acid sequence alignments of CDRs of PSCA mAbs 1G8,4A10 and 2H9.

[0077]FIG. 62. Photographs showing PSCA protein expression in normalbladder and various bladder carcinoma tissues using immunohistochemicalstaining of paraffin-embedded samples with PSCA mAb 1G8.

[0078]FIG. 63. Northern blot analysis of PSCA expression in severalpancreatic cancer cells lines. Northern blot analysis of PSCA expressionin normal prostate and several prostate cancer xenografts are shownalongside for comparison. RNA levels between all samples werenormalized.

[0079]FIG. 64. Western blot analysis of PSCA protein expression inprostate and pancreatic cancer cell line using PSCA mab 1G8.

[0080]FIG. 65. PSCA mAbs exert growth inhibitory effect through PSCAprotein. The growth inhibitory effect of PSCA mAb 1G8 on LAPC-9 and PC-3prostate tumors is compared, showing no effect on PC-3 tumors, which donot express PSCA antigen, but significant growth inhibition in LAPC-9tumors, which do express PSCA antigen. See Examples 18-C1, -C3 fordetails.

[0081]FIG. 66. Growth inhibition of established LAPC-9 (AD) orthotopictumors by the anti-PSCA mAb 1G8. (A) Mice having low levels of serumPSA. Two mg of 1G8 was administered to these mice on days 10, 13, and15, followed by one mg on days 17, 20, 22, 25, 27, 29, 34, 41, and 49 asindicated by the arrows. (B) Mice having moderate levels of serum PSA.One mg of 1G8 was administered on days 12, 13, 14, 19, 20, 22, 25, 27,29, and 33 as indicated by the arrows.

[0082]FIG. 67. Treatment with the anti-PSCA mAb, 1G8, increases survivalof mice bearing established LAPC-9 (AD) orthotopic tumors. (A) The micein FIG. 66 A, which were treated with 1G8, exhibited an increase insurvival compared to mice treated with PBS. (B) The mice in FIG. 66 B,which were treated with 1G8, exhibited an increase in survival comparedto mice treated with PBS.

[0083]FIG. 68. Growth inhibition of established LAPC-9 AD orthotopictumors by, the anti-PSCA mAb 3C5. (A) One mg of 3C5 was administered totumor-bearing mice on days 6, 8, 10, 13, 15, 17, 20, 22, 24, and 29 asindicated by the arrows. The mice were bled on the days indicated on theX-axis for PSA determinations. (B) Two mg of 3C5 was administered totumor-bearing mice on days 9, 12, and 15, followed by one mg on days 18,20, 22, 25, 27, and 29 as indicated by the arrows. The mice were bled onthe days indicated on the X-axis for PSA determinations.

[0084]FIG. 69. Treatment with the anti-PSCA mAb, 3C5, increases survivalof mice bearing LAPC-9 AD orthotopic tumors. (A) The mice in FIG. 68 A,which were treated with 3C5, exhibited an increase in survival comparedto mice treated with PBS. There were 4 mice in the PBS-treated group and5 mice in the 3C5-treated group. (B) The mice in FIG. 68 B, which weretreated with 3C5, exhibited an increase in survival compared to micetreated with PBS. There were 6 mice in both the PBS-treated and3C5-treated groups.

[0085]FIG. 70. Growth inhibition of established PC3-PSCA tumors by 1G8alone or in combination with doxorubicin. One mg of 1G8 was administeredto tumor-bearing mice on days 9, 11, 14, 16, 18, 21, 23, 25, and 28 asindicated by the arrows. Twenty-five micrograms of doxorubicin wasadministered on days 9, 16, and 23 as indicated by the (•)symbol.

[0086]FIG. 71. Anti-PSCA antibody administered to tumor-bearing micecirculates and targets tumors expressing PSCA A) Immunohistochemistry ofa tumor explant from a mouse, bearing an established PSCA-expressingtumor, treated with 3C5. B) Immunohistochemistry of a tumor explant froma mouse, bearing an established PSCA-expressing tumor, treated withmouse IgG.

[0087]FIG. 72. Anti-PSCA antibody administered to a tumor-bearing mousecirculates and targets tumors expressing PSCA. A Western blot analysisof tumor lysates from tumors explanted from mice described in FIG. 71,probed with goat anti-mouse IgG-HRP antibody.

[0088]FIG. 73. Anti-PSCA antibody administered to a tumor-bearing mousecirculates and targets tumors expressing PSCA. A Western blot analysisof tumor lysates from tumors explanted from mice bearing establishedPSCA-expressing tumors, treated with 1G8. The blot was probed with goatanti-mouse IgG-HRP antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0089] The present invention relates to Prostate Stem Cell Antigen(hereinafter “PSCA”). PSCA is a novel, glycosylphosphatidylinositol(GPI)-anchored cell surface antigen which is expressed in normal cellssuch prostate cells, urothelium, renal collecting ducts, colonicneuroendocrine cells, placenta, normal bladder and urethral transitionalepithelial cells (FIG. 16). PSCA, in addition to normal cells, is alsooverexpressed by both androgen-dependent and androgen-independentprostate cancer cells (FIG. 9-11), prostate cancer metastases to bone(FIG. 20-24 and 26-32), bladder carcinomas (FIG. 6, 25 and 62), andpancreatic carcinomas (FIGS. 63 and 64). The expression of PSCA incancer, e.g., prostate cancer and bladder cancer, appears to correlatewith increasing grade. Further, overexpression of PSCA (i.e. higherexpression than found in normal cells) in patients suffering fromcancer, e.g., prostate cancer, appears to be indicative of poorprognosis.

[0090] PSCA mRNA is also expressed by a subset of basal cells in normalprostate. The basal cell epithelium is believed to contain theprogenitor cells for the terminally differentiated secretory cells(Bonkhoff et al., 1994, Prostate 24: 114-118). Recent studies usingcytokeratin markers suggest that the basal cell epithelium contains atleast two distinct cellular subpopulations, one expressing cytokeratins5 and 14 and the other cytokeratins 5, 8 and 18 (Bonkhoff and Remberger,1996, Prostate 28: 98-106). The finding that PSCA is expressed by only asubset of basal cells suggests that PSCA may be a marker for one ofthese two basal cell lineages.

[0091] The biological function of PSCA is unknown. The Ly-6 gene familyis involved in diverse cellular functions, including signal transductionand cell-cell adhesion. Signaling through SCA-2 has been demonstrated toprevent apoptosis in immature thymocytes (Noda et al., 1996, J. Exp.Med. 183: 2355-2360). Thy-1 is involved in T cell activation andtransmits signals through src-like tyrosine kinases (Thomas et al.,1992, J. Biol. Chem. 267: 12317-12322). Ly-6 genes have been implicatedboth in tumorigenesis and in homotypic cell adhesion (Bamezai and Rock,1995, Proc. Natl. Acad. Sci. USA 92: 4294-4298; Katz et al., 1994, Int.J. Cancer 59: 684-691; Brakenhoffet al., 1995, J. Cell Biol. 129:1677-1689). Based on its restricted expression in basal cells and itshomology to Sca-2, we hypothesize that PSCA may play a role instem/progenitor cell functions such as self-renewal (anti-apoptosis)and/or proliferation.

[0092] PSCA is highly conserved in mice and humans. The identificationof a conserved gene which is predominantly restricted to prostatesupports the hypothesis that PSCA may play an important role in normalprostate development.

[0093] In its various aspects, as described in detail below, the presentinvention provides PSCA proteins, antibodies, nucleic acid molecules,recombinant DNA molecules, transformed host cells, generation methods,assays, immunotherapeutic methods, transgenic animals, immunological andnucleic acid-based assays, and compositions.

[0094] PSCA Proteins

[0095] One aspect of the invention provides various PSCA proteins andpeptide fragments thereof As used herein, PSCA refers to a protein thathas the amino acid sequence of human PSCA as provided in FIGS. 1B and 3,the amino acid sequence of the murine PSCA homologue as provided in FIG.3, or the amino acid sequence of other mammalian PSCA homologues, aswell as allelic variants and conservative substitution mutants of theseproteins that have PSCA activity. The PSCA proteins of the inventioninclude the specifically identified and characterized variants hereindescribed, as well as allelic variants, conservative substitutionvariants and homologs that can be isolated/generated and characterizedwithout undue experimentation following the methods outlined below. Forthe sake of convenience, all PSCA proteins will be collectively referredto as the PSCA proteins, the proteins of the invention, or PSCA.

[0096] The term “PSCA” includes all naturally occurring allelicvariants, isoforms, and precursors of human PSCA as provided in FIGS. 1Band 3 and murine PSCA as provided in FIG. 3. In general, for example,naturally occurring allelic variants of human PSCA will sharesignificant homology (e.g., 70-90%) to the PSCA amino acid sequenceprovided in FIGS. 1B and 3. Allelic variants, though possessing aslightly different amino acid sequence, may be expressed on the surfaceof prostate cells as a GPI linked protein or may be secreted or shed.Typically, allelic variants of the PSCA protein will containconservative amino acid substitutions from the PSCA sequence hereindescribed or will contain a substitution of an amino acid from acorresponding position in a PSCA homologue such as, for example, themurine PSCA homologue described herein.

[0097] One class of PSCA allelic variants will be proteins that share ahigh degree of homology with at least a small region of the PSCA aminoacid sequences presented in FIGS. 1B and 3, but will further contain aradical departure from the sequence, such as a non-conservativesubstitution, truncation, insertion or frame shift. Such alleles aretermed mutant alleles of PSCA and represent proteins that typically donot perform the same biological functions.

[0098] Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in Which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

[0099] The amino acid sequence of human PSCA protein is provided inFIGS. 1B and 3. Human PSCA is comprised of a single subunit of 123 aminoacids and contains an amino-terminal signal sequence, a carboxy-terminalGPI-anchoring sequence, and multiple N-glycosylation sites. PSCA shows30% homology to stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly-6gene family, a group of cell surface proteins which mark the earliestphases of hematopoetic development. The amino acid sequence of a murinePSCA homologue is shown in FIG. 3. Murine PSCA is a single subunitprotein of 123 amino acids having approximately 70% homology to humanPSCA and similar structural organization.

[0100] PSCA proteins may be embodied in many forms, preferably in anisolated form. As used herein, a protein is said to be isolated whenphysical, mechanical or chemical methods are employed to remove the PSCAprotein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated PSCA protein. A purified PSCA proteinmolecule will be substantially free of other proteins or molecules thatimpair the binding of PSCA to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of the PSCA protein include a purified PSCA protein and afunctional, soluble PSCA protein. One example of a functional solublePSCA protein has the amino acid sequence shown in FIG. 1B or a fragmentthereof. In one form, such functional, soluble PSCA proteins orfragments thereof retain the ability to bind antibody or other ligand.

[0101] The invention also provides peptides comprising biologicallyactive fragments of the human and murine PSCA amino acid sequences shownin FIGS. 1B and 3. For example, the invention provides a peptidefragment having the amino acid sequence TARIRAVGLLTVISK, a peptidefragment having the amino acid sequence VDDSQDYYVGKK, andSLNCVDDSQDYYVGK.

[0102] The peptides of the invention exhibit properties of PSCA, such asthe ability to elicit the generation of antibodies that specificallybind an epitope associated with PSCA. Such peptide fragments of the PSCAproteins can be generated using standard peptide synthesis technologyand the amino acid sequences of the human or murine PSCA proteinsdisclosed herein. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a fragment of the PSCAprotein. In this regard, the PSCA-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of PSCA.

[0103] As discussed below, peptide fragments of PSCA are particularlyuseful in: generating domain specific antibodies; identifying agentsthat bind to PSCA or a PSCA domain; identifying cellular factors thatbind to PSCA or a PSCA domain; and isolating homologs or other allelicforms of human PSCA. PSCA peptides containing particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Asexamples, hydrophobicity and Chou-Fasman plots of human PSCA areprovided in FIGS. 4 and 5 respectively. Fragments containing suchresidues are particularly useful in generating subunit specificanti-PSCA antibodies or in identifying cellular factors that bind toPSCA.

[0104] Various regions of the PSCA protein can bind to anti-PSCAantibodies. The regions of the PSCA protein may include, for example,the N-terminal region, middle region, and C-terminal region (Example 18,FIG. 49). The N-terminal region includes any portion of the PSCA proteinencompassed by amino acid residues 2-50, preferably residues 18-50. Themiddle region includes any portion of the PSCA protein encompassed byamino acid residues 46-109, preferably residues 46-98. The C-terminalregion includes any portion of the PSCA protein encompassed by aminoacid residues 85-123, preferably residues 85-98.

[0105] The PSCA proteins of the invention may be useful for a variety ofpurposes, including but not limited to their use as diagnostic and/orprognostic markers of prostate cancer, the ability to elicit thegeneration of antibodies, and as targets for various therapeuticmodalities, as further described below. PSCA proteins may also be usedto identify and isolate ligands and other agents that bind to PSCA. Inthe normal prostate, PSCA is expressed exclusively in a subset of basalcells, suggesting that PSCA may be used as a marker for a specific celllineage within basal epithelium. In addition, the results herein suggestthat this set of basal cells represent the target of neoplastictransformation. Accordingly for example, therapeutic strategies designedto eliminate or modulate the molecular factors responsible fortransformation may be specifically directed to this population of cellsvia the PSCA cell surface protein.

[0106] PSCA Antibodies

[0107] The invention further provides antibodies (e.g., polyclonal,monoclonal, chimeric, and humanized antibodies) that bind to PSCA. Themost preferred antibodies will selectively bind to PSCA and will notbind (or will bind weakly) to non-PSCA proteins. The most preferredantibodies will specifically bind to PSCA. It is intended that the term“specifically bind” means that the antibody predominantly binds to PSCA.Anti-PSCA antibodies that are particularly contemplated includemonoclonal and polyclonal antibodies as well as fragments thereof (e.g.,recombinant proteins) containing the antigen binding domain and/or oneor more complement determining regions of these antibodies. Theseantibodies can be from any source, e.g., rat, dog, cat, pig, horse,mouse or human.

[0108] In one embodiment, the PSCA antibodies specifically bind to theextracellular domain of a PSCA protein, e.g., on the cell surface ofprostate cancer cells from primary lesions and prostate cancer bonemetastases. It is intended that the term “extracellular domain” meansany portion of the PSCA protein which is exterior to the plasma membraneof the cell. In other embodiments, the PSCA antibodies specifically bindto other domains of a PSCA protein or precursor (such as a portion ofthe N-terminal region, the middle region, or the C-terminal region; FIG.49). As will be understood by those skilled in the art, the regions orepitopes of a PSCA protein to which an antibody is directed may varywith the intended application. For example, antibodies intended for usein an immunoassay for the detection of membrane-bound PSCA on viableprostate cancer cells should be directed to an accessible epitope onmembrane-bound PSCA. Examples of such antibodies are described theExamples which follow. Antibodies that recognize other epitopes may beuseful for the identification of PSCA within damaged or dying cells, forthe detection of secreted PSCA proteins or fragments thereof. Theinvention also encompasses antibody fragments that specificallyrecognize a PSCA protein. As used herein, an antibody fragment isdefined as at least a portion of the variable region of theimmunoglobulin molecule that binds to its target, i.e., the antigenbinding region. Some of the constant region of the immunoglobulin may beincluded.

[0109] For example, the overexpression of PSCA in bothandrogen-dependent and androgen-independent prostate cancer cells, andthe cell surface location of PSCA represent characteristics of anexcellent marker for screening, diagnosis, prognosis, and follow-upassays and imaging methods. In addition, these characteristics indicatethat PSCA may be an excellent target for therapeutic methods such astargeted antibody therapy, immunotherapy, and gene therapy.

[0110] PSCA antibodies of the invention may be particularly useful indiagnostic assays, imaging methodologies, and therapeutic methods in themanagement of prostate cancer. The invention provides variousimmunological assays useful for the detection of PSCA proteins and forthe diagnosis of prostate cancer. Such assays generally comprise one ormore PSCA antibodies capable of recognizing and binding a PSCA protein,and include various immunological assay formats well known in the art,including but not limited to various types of precipitation,agglutination, complement fixation, radioimmunoassays (RIA),enzyme-linked immunosorbent assays (ELISA), enzyme-linkedimmunofluorescent assays (ELIFA) (H. Liu et al. Cancer Research 58:4055-4060 (1998), immunohistochemical analysis and the like. Inaddition, immunological imaging methods capable of detecting prostatecancer are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled PSCA antibodies. Suchassays may be clinically useful in the detection, monitoring, andprognosis of prostate cancer.

[0111] In one embodiment, PSCA antibodies and fragments thereof (e.g.,Fv, Fab′, F(ab′)2) are used for detecting the presence of a prostatecancer, bladder carcinoma, pancreatic carcinoma, bone metastases ofprostate cancer, PIN, or basal epithelial cell expressing a PSCAprotein. The presence of such PSCA positive (+) cells within variousbiological samples, including serum, prostate and other tissue biopsyspecimens, other tissues such as bone, urine, etc., may be detected withPSCA antibodies. In addition, PSCA antibodies may be used in variousimaging methodologies, such as immunoscintigraphy with Induim-111 (orother isotope) conjugated antibody. For example, an imaging protocolsimilar to the one recently described using an In-111 conjugatedanti-PSMA antibody may be used to detect recurrent and metastaticprostate carcinomas (Sodee et al., 1997, Clin Nuc Med 21: 759-766). Inrelation to other markers of prostate cancer, such as PSMA for example,PSCA may be particularly useful for targeting androgen independentprostate cancer cells. PSCA antibodies may also be used therapeuticallyto inhibit PSCA function.

[0112] PSCA antibodies may also be used in methods for purifying PSCAproteins and peptides and for isolating PSCA homologues and relatedmolecules. For example, in one embodiment, the method of purifying aPSCA protein comprises incubating a PSCA antibody, which has beencoupled to a solid matrix, with a lysate or other solution containingPSCA under conditions which permit the PSCA antibody to bind to PSCA;washing the solid matrix to eliminate impurities; and eluting the PSCAfrom the coupled antibody. Additionally, PSCA antibodies may be used toisolate PSCA positive cells using cell sorting and purificationtechniques. The presence of PSCA on prostate tumor cells (alone or incombination with other cell surface markers) may be used to distinguishand isolate human prostate cancer cells from other cells. In particular,PSCA antibodies may be used to isolate prostate cancer cells fromxenograft tumor tissue, from cells in culture, etc., usingantibody-based cell sorting or affinity purification techniques. Otheruses of the PSCA antibodies of the invention include generatinganti-idiotypic antibodies that mimic the PSCA protein, e.g., amonoclonal anti-idiotypic antibody reactive with an idiotype on any ofthe monoclonal antibodies of the invention such as 1G8, 2A2, 2H9, 3C5,3E6, 3G3, and 4A10.

[0113] The ability to generate large quantities of relatively pure humanPSCA positive prostate cancer cells which can be grown in tissue cultureor as xenograft tumors in animal models (e.g., SCID or other immunedeficient mice) provides many advantages, including, for example,permitting the evaluation of various transgenes or candidate therapeuticcompounds on the growth or other phenotypic characteristics of arelatively homogeneous population of prostate cancer cells.Additionally, this feature of the invention also permits the isolationof highly enriched preparations of human PSCA positive prostate cancerspecific nucleic acids in quantities sufficient for various molecularmanipulations. For example, large quantities of such nucleic acidpreparations will assist in the identification of rare genes withbiological relevance to prostate cancer disease progression.

[0114] Another valuable application of this aspect of the invention isthe ability to isolate, analyze and experiment with relatively purepreparations of viable PSCA positive prostate tumor cells cloned fromindividual patients with locally advanced or metastatic disease. In thisway, for example, an individual patient's prostate cancer cells may beexpanded from a limited biopsy sample and then tested for the presenceof diagnostic and prognostic genes, proteins, chromosomal aberrations,gene expression profiles, or other relevant genotypic and phenotypiccharacteristics, without the potentially confounding variable ofcontaminating cells. In addition, such cells may be evaluated forneoplastic aggressiveness and metastatic potential in animal models.Similarly, patient-specific prostate cancer vaccines and cellularimmunotherapeutics may be created from such cell preparations.

[0115] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies may be prepared by immunizing asuitable mammalian host using a PSCA protein, peptide, or fragment, inisolated or immunoconjugated form (Harlow, Antibodies, Cold SpringHarbor Press, N.Y. (1989)). In addition, fusion proteins of PSCA mayalso be used, such as a PSCA GST-fusion protein. Cells expressing oroverexpressing PSCA may also be used for immunizations. Similarly, anycell engineered to express PSCA may be used. This strategy may result inthe production of monoclonal antibodies with enhanced capacities forrecognizing endogenous PSCA. For example, using standard technologiesdescribed in Example 5 and standard hybridoma protocols (Harlow andLane, 1988, Antibodies: A Laboratory Manual. (Cold Spring HarborPress)), hybridomas producing monoclonal antibodies designated 1G8 (ATCCNo. HB-12612), 2A2 (ATCC No. HB-12613), 2H9 (ATCC No. HB-12614), 3C5(ATCC No. HB-12616), 3E6 (ATCC No. HB-12618), and 3G3 (ATCC No.HB-12615), 4A10 (ATCC No. HB-12617) were generated. These antibody weredeposited on Dec. 11, 1998 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

[0116] Chimeric antibodies of the invention are immunoglobulin moleculesthat comprise a human and non-human portion. The antigen combiningregion (variable region) of a chimeric antibody can be derived from anon-human source (e.g. murine) and the constant region of the chimericantibody which confers biological effector function to theimmunoglobulin can be derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule.

[0117] In general, the procedures used to produce chimeric antibodiescan involve the following steps:

[0118] a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains or simply asthe V or variable region) may be in either the cDNA or genomic form;

[0119] b) cloning the gene segments encoding the constant region ordesired part thereof;

[0120] c) ligating the variable region with the constant region so thatthe complete chimeric antibody is encoded in a form that can betranscribed and translated;

[0121] d) ligating this construct into a vector containing a selectablemarker and gene control regions such as promoters, enhancers and poly(A)addition signals;

[0122] e) amplifying this construct in bacteria;

[0123] f) introducing this DNA into eukaryotic cells (transfection) mostoften mammalian lymphocytes;

[0124] g) selecting for cells expressing the selectable marker;

[0125] h) screening for cells expressing the desired chimeric antibody;and

[0126] k) testing the antibody for appropriate binding specificity andeffector functions.

[0127] Antibodies of several distinct antigen binding specificities havebeen manipulated by these protocols to produce chimeric proteins [e.g.anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumorantigens: Sahagan et al., J. Immunol. 137:1066 (1986)]. Likewise,several different effector functions have been achieved by lining newsequences to those encoding the antigen binding region. Some of theseinclude enzymes [Neuberger et al., Nature 312:604 (1984)],immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain [Sharon et al., Nature 309:364(1984); Tan et al., J. Immunol. 135:3565-3567 (1985)]. Additionally,procedures for modifying antibody molecules and for producing chimericantibody molecules using homologous recombination to target genemodification have been described (Fell et al., Proc. Natl. Acad. Sci.USA 86:8507-8511 (1989)).

[0128] These antibodies are capable of binding to PSCA, e.g., on thecell surface of prostate cancer cells, thereby confirming the cellsurface localization of PSCA. Because these mAbs recognize epitopes onthe exterior of the cell surface, they have utility for prostate cancerdiagnosis and therapy. For example, these mAbs were used to locate sitesof metastatic disease (Example 6). Another possibility is that they maybe used (e.g., systemically) to target prostate cancer cellstherapeutically when used alone or conjugated to a radioisotope or othertoxin.

[0129] PSCA mAbs stain the cell surface in a punctate manner (seeExample 5), suggesting that PSCA may be localized to specific regions ofthe cell surface. GPI-anchored proteins are known to cluster indetergent-insoluble glycolipid-enriched microdomains (DIGS) oft the cellsurface. These microdomains, which include caveolae andshingolipid-cholesterol rafts, are believed to play critical roles insignal transduction and molecular transport. Thy-1, a homologue of PSCA,has previously been shown to transmit signals to src kinases throughinteraction in lipid-microdomains. Subcellular fractionation experimentsin our laboratory confirm the presence of PSCA in DIGS.

[0130] Additionally, some of the antibodies of the invention areinternalizing antibodies, i.e., the antibodies are internalized into thecell upon or after binding. It is intended that the term “internalize”means that the antibody is taken into the cell. Further, some of theantibodies induce inhibition of PSCA positive cancer cell growth.

[0131] A characterization of these antibodies, e.g., in prostate cancerspecimens, demonstrates that PSCA protein is overexpressed in prostatecancers relative to normal cells and its expression appears to beupregulated during prostate cancer progression and metastasis. Theseantibodies are useful in studies of PSCA biology and function, as wellas in vivo targeting of PSCA associated cancers, including, withoutlimitation, human prostate cancer, prostate cancer metastases to bone,bladder carcinomas, and pancreatic carcinomas.

[0132] PSCA mAbs which specifically recognize and bind to theextracellular domain of the PSCA protein are described herein. Some ofthese have been shown to bind to native PSCA as expressed on the cellsurface and some have been shown to inhibit the in vivo growth ofprostate tumor cells.

[0133] The amino acid sequence of PSCA presented herein may be used toselect specific regions of the PSCA protein for generating antibodies.For example, hydrophobicity and hydrophilicity analyses of the PSCAamino acid sequence may be used to identify hydrophilic regions in thePSCA structure. Regions of the PSCA protein that show immunogenicstructure, as well as other regions and domains, can readily beidentified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Fragments containing these residues areparticularly suited in generating specific classes of anti-PSCAantibodies. Particularly useful fragments include, but are not limitedto, the sequences TARIRAVGLLTVISK and SLNCVDDSQDYYVGK.

[0134] As described in Example 2, below, a rabbit polyclonal antibodywas generated against the former fragment, prepared as a syntheticpeptide, and affinity purified using a PSCA-glutathione S transferasefusion protein. Recognition of PSCA by this antibody was established byimmunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. This antibody alsoidentified the cell surface expression of PSCA in PSCA-transfected 293Tand LAPC-4 cells using fluorescence activated cell sorting (FACS).

[0135] Additionally, a sheep polyclonal antibody was generated againstthe latter fragment, prepared as a synthetic peptide, and affinitypurified using a peptide Affi-gel column (also by the method of Example2). Recognition of PSCA by this antibody was established by immunoblotand immunoprecipitation analysis of extracts of LNCaP cells transfectedwith PSCA. This antibody also identified the cell surface expression ofPSCA in PSCA-transfected LNCAP cells using fluorescence activated cellsorting (FACS) and immunohistochemistry analysis.

[0136] Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation using, for example, carbodiimidereagents may be used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., may be effective.Administration of a PSCA immunogen is conducted generally by injectionover a suitable time period and with use of a suitable adjuvant, as isgenerally understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

[0137] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions,monoclonal antibody preparations are preferred. Immortalized cell lineswhich secrete a desired monoclonal antibody may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the PSCA protein or PSCAfragment. When the appropriate immortalized cell culture secreting thedesired antibody is identified, the cells can be cultured either invitro or by production in ascites fluid.

[0138] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonal antibodies of the invention or the polyclonal antisera (e.g.,Fab, F(ab′)₂, Fv fragments, fusion proteins) which contain theimmunologically significant portion (i.e., a portion that recognizes andbinds PSCA) can be used as antagonists, as well as the intactantibodies. Humanized antibodies directed against PSCA is also useful.As used herein, a humanized PSCA antibody is an immunoglobulin moleculewhich is capable of binding to PSCA and which comprises a FR regionhaving substantially the amino acid sequence of a human immunoglobulinand a CDR having substantially the amino acid sequence of non-humanimmunoglobulin or a sequence engineered to bind PSCA. Methods forhumanizing murine and other non-human antibodies by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmnan et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

[0139] Use of immunologically reactive fragments, such as the Fab, Fab′,or F(ab′)₂ fragments is often preferable, especially in a therapeuticcontext, as these fragments are generally less immunogenic than thewhole immunoglobulin. Further, bi-specific antibodies specific for twoor more epitopes may be generated using methods generally known in theart. Further, antibody effector functions may be modified so as toenhance the therapeutic effect of PSCA antibodies on cancers. Forexample, cysteine residues may be engineered into the Fc region,permitting the formation of interchain disulfide bonds and thegeneration of homodimers which may have enhanced capacities forinternalization, ADCC′ and/or complement-mediated cell killing (see, forexample, Caron et al., 1992, J. Exp. Med. 176: 1191-1195; Shopes, 1992,J. Immunol. 148: 2918-2922). Homodimeric antibodies may also begenerated by cross-linking techniques known in the art (e.g., Wolff etal., Cancer Res. 53: 2560-2565). The invention also providespharmaceutical compositions having the monoclonal antibodies oranti-idiotypic monoclonal antibodies of the invention.

[0140] The generation of monoclonal antibodies (mAbs) capable of bindingto cell surface PSCA are described in Example 5. Epitope mapping ofthese mAbs indicates that they recognize different epitopes on the PSCAprotein. For example, one recognizes an epitope within thecarboxy-terminal region and the other recognizing an epitope within theamino-terminal region. Such PSCA antibodies may be particularly wellsuited to use in a sandwich-formatted ELISA, given their differingepitope binding characteristics.

[0141] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the PSCA protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin. Theinvention includes an antibody, e.g., a monoclonal antibody whichcompetitively inhibits the immunospecific binding of any of themonoclonal antibodies of the invention to PSCA.

[0142] Alternatively, methods for producing fully human monoclonalantibodies, include phage display and transgenic methods, are known andmay be used for the generation of human mAbs (for review, see Vaughan etal., 1998, Nature Biotechnology 16: 535-539). For example, fully humananti-PSCA monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display)(Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. Ed.), Nottingham Academic, pp45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human anti-PSCA monoclonal antibodiesmay also be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Jakobovits et al., published Dec. 3, 1997 (see also,Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614). This methodavoids the in vitro manipulation required with phage display technologyand efficiently produces high affinity authentic human antibodies.

[0143] Reactivity of anti-PSCA mAbs against the target antigen may beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,PSCA proteins, peptides, PSCA-expressing cells or extracts thereof.Anti-PSCA mAbs may also be characterized in various in vitro assays,including complement-mediated tumor cell lysis, antibody-dependent cellcytotoxicity (ADCC), antibody-dependent macrophage-mediated cytotoxicity(ADMMC), tumor cell proliferation, etc. Examples of such in vitro assaysare presented in Example 19, infra.

[0144] The antibody or fragment thereof of the invention may becytostatic to the cell, to which it binds. It is intended that the term“cytostatic” means that the antibody can inhibit growth, but notnecessarily kill, PSCA-positive cells.

[0145] The antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule, such as atherapeutic agent (e.g., a cytotoxic agent) thereby resulting in animmunoconjugate. For example, the therapeutic agent includes, but is notlimited to, an anti-tumor drug, a toxin, a radioactive agent, acytokine, a second antibody or an enzyme. Further, the inventionprovides an embodiment wherein the antibody of the invention is linkedto an enzyme that converts a prodrug into a cytotoxic drug.

[0146] The immunoconjugate can be used for targeting the second moleculeto a PSCA positive cell (Vitetta, E. S. et al., 1993, Immunotoxintherapy, in DeVita, Jr., V. T. et al., eds, Cancer: Principles andPractice of Oncology, 4th ed., J. B. Lippincott Co., Philadelphia,2624-2636).

[0147] Examples of cytotoxic agents include, but are not limited toricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduimbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin Achain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, maytansinoids, and glucocorticoid and other chemotherapeuticagents, as well as radioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and¹⁸⁶Re. Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. Antibodies mayalso be conjugated to an anti-cancer pro-drug activating enzyme capableof converting the pro-drug to its active form. See, for examples, U.S.Patent No. 4,975,287.

[0148] Additionally, the recombinant protein of the invention comprisingthe antigen-binding region of any of the monoclonal antibodies of theinvention can be used to treat cancer. In such a situation, theantigen-binding region of the recombinant protein is joined to at leasta functionally active portion of a second protein having therapeuticactivity. The second protein can include, but is not limited to, anenzyme, lymphokine, oncostatin or toxin. Suitable toxins include thosedescribed above.

[0149] Techniques for conjugating or joining therapeutic agents toantibodies are well known (see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); and Thorpe et al., “The Preparation AndCytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev.,62:119-58 (1982)). The use of PSCA antibodies as therapeutic agents isfurther described in the subsection “PROSTATE CANCER IMMUNOTHERAPY”below.

[0150] PSCA-Encoding Nucleic Aced Molecules

[0151] Another aspect of the invention provides various nucleic acidmolecules encoding PSCA proteins and fragments thereof, preferably inisolated form, including DNA, RNA, DNA/RNA hybrid, and relatedmolecules, nucleic acid molecules complementary to the

[0152] PSCA coding sequence or a part thereof, and those which hybridizeto the PSCA gene or to PSCA-encoding nucleic acids. Particularlypreferred nucleic acid molecules will have a nucleotide sequencesubstantially identical to or complementary to the human or murine DNAsequences herein disclosed. Specifically contemplated are genomic DNA,cDNAs, ribozymes, and antisense molecules, as well as nucleic acidsbased on an alternative backbone or including alternative bases, whetherderived from natural sources or synthesized.

[0153] For example, antisense molecules can be RNAs or other molecules,including peptide nucleic acids (PNAs) or non-nucleic acid moleculessuch as phosphorothioate derivatives, that specifically bind DNA or RNAin a base pair-dependent manner. A skilled artisan can readily obtainthese classes of nucleic acid molecules using the herein described PSCAsequences. For convenience, PSCA-encoding nucleic acid molecules will bereferred to herein as PSCA-encoding nucleic acid molecules, PSCA genes,or PSCA sequences.

[0154] The nucleotide sequence of a cDNA encoding one allelic form ofhuman PSCA is provided in FIG. 1A. The nucleotide sequence of a cDNAencoding a murine PSCA homologue (“murine PSCA”) is provided in FIG. 2.Genomic clones of human and murine PSCA have also been isolated, asdescribed in Example 4. Both the human and murine genomic clones containthree exons encoding the translated and 3′ untranslated regions of thePSCA gene. A fourth exon encoding a 5′ untranslated region is presumedto exist based on PSCA's homology to other members of the Ly-6 and Thy-1gene families (FIG. 8).

[0155] The human PSCA gene maps to chromosome 8q24.2. Human stem cellantigen 2 (RIG-E), as well as one other recently identified human Ly-6homologue (E48) are also localized to this region, suggesting that alarge family of related genes may exist at this locus (Brakenhoff etal., 1995, supra; Mao et al., 1996, Proc. Natl. Acad. Sci. USA 93:5910-5914). Intriguingly, chromosome 8q has been reported to be a regionof allelic gain and amplification in a majority of advanced andrecurrent prostate cancers (Cher et al., 1994, Genes Chrom. Cancer 11:153-162). c-myc localizes proximal to PSCA at chromosome 8q24 and extracopies of c-myc (either through allelic gain or amplification) have beenfound in 68% of primary prostate tumors and 96% of metastases (Jenkinset al., 1997, Cancer Res. 57: 524-531).

[0156] Embodiments of the PSCA-encoding nucleic acid molecules of theinvention include primers, which allow the specific amplification ofnucleic acid molecules of the invention or of any specific partsthereof, and probes that selectively or specifically hybridize tonucleic acid molecules of the invention or to any part thereof. Thenucleic acid probes can be labeled with a detectable marker. Examples ofa detectable marker include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator or an enzyme. Such labeled probes can be usedto diagnosis the presence of a PSCA protein as a means for diagnosingcell expressing a PSCA protein. Technologies for generating DNA and RNAprobes are well-known.

[0157] As used herein, a nucleic acid molecule is said to be “isolated”when the nucleic acid molecule is substantially separated fromcontaminant nucleic acid molecules that encode polypeptides other thanPSCA. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated PSCA-encoding nucleic acid molecule.

[0158] The invention further provides fragments of the PSCA-encodingnucleic acid molecules of the present invention. As used herein, afragment of a PSCA-encoding nucleic acid molecule refers to a smallportion of the entire PSCA-encoding sequence. The size of the fragmentwill be determined by its intended use.

[0159] For example, if the fragment is chosen so as to encode an activeportion of the PSCA protein, such an active domain, effector bindingsite or GPI binding domain, then the fragment will need to be largeenough to encode the functional region(s) of the PSCA protein. If thefragment is to be used as a nucleic acid probe or PCR primer, then thefragment length is chosen so as to obtain a relatively small number offalse positives during probing/priming.

[0160] Fragments of human PSCA that are particularly useful as selectivehybridization probes or PCR primers can be readily identified from theentire PSCA sequence using art-known methods. One set of PCR primersthat are useful for RT-PCR analysis comprise 5′ -TGCTTGCCCTGTTGATGGCAG-and 3′ -CCAGAGCAGCAGGCCGAGTGCA-.

[0161] Methods for Isolating other PSCA-Encoding Nucleic Acid Molucules

[0162] The PSCA-encoding nucleic acid molecules described herein enablethe isolation of PSCA homologues, alternatively sliced isoforms, allelicvariants, and mutant forms of the PSCA protein as well as their codingand gene sequences. The most preferred source of PSCA homologs aremammalian organisms.

[0163] For example, a portion of the PSCA-encoding sequence hereindescribed can be synthesized and used as a probe to retrieve DNAencoding a member of the PSCA family of proteins from organisms otherthan human, allelic variants of the human PSCA protein herein described,and genomic sequence containing the PSCA gene. Oligomers containingapproximately 18-20 nucleotides (encoding about a 6-7 amino acidstretch) are prepared and used to screen genomic DNA or cDNA librariesto obtain hybridization under stringent conditions or conditions ofsufficient stringency to eliminate an undue level of false positives. Ina particular embodiment, cDNA encoding human PSCA was used to isolate afull length cDNA encoding the murine PSCA homologue as described inExample 3 herein. The murine clone encodes a protein with 70% amino acididentity to human PSCA.

[0164] In addition, the amino acid sequence of the human PSCA proteinmay be used to generate antibody probes to screen expression librariesprepared from cells. Typically, polyclonal antiserum from mammals suchas rabbits immunized with the purified protein (as described below) ormonoclonal antibodies can be used to probe an expression library,prepared from a target organism, to obtain the appropriate codingsequence for a PSCA homologue. The cloned cDNA sequence can be expressedas a fusion protein, expressed directly using its own control sequences,or expressed by constructing an expression cassette using controlsequences appropriate to the particular host used for expression of theenzyme.

[0165] Genomic clones containing PSCA genes may be obtained usingmolecular cloning methods well known in the art. In one embodiment, ahuman genomic clone of approximately 14kb containing exons 14 of thePSCA gene was obtained by screening a lambda phage library with a humanPSCA cDNA probe, as more completely described in Example 4 herein. Inanother embodiment, a genomic clone of approximately 10kb containing themurine PSCA gene was obtained by screening a murine BAC (bacterialartificial chromosome) library with a murine PSCA cDNA (also describedin Example 4).

[0166] Additionally, pairs of oligonucleotide primers can be preparedfor use in a polymerase chain reaction (PCR) to selectivelyamplify/clone a PSCA-encoding nucleic acid molecule, or fragmentthereof. A PCR denature/anneal/extend cycle for using such PCR primersis well known in the art and can readily be adapted for use in isolatingother PSCA-encoding nucleic acid molecules. Regions of the human PSCAgene that are particularly well suited for use as a probe or as primerscan be readily identified.

[0167] Non-human homologues of PSCA, naturally occurring allelicvariants of PSCA and genomic PSCA sequences will share a high degree ofhomology to the human PSCA sequences herein described. In general, suchnucleic acid molecules will hybridize to the human PSCA sequence understringent conditions. Such sequences will typically contain at least 70%homology, preferably at least 80%, most preferably at least 90% homologyto the human PSCA sequence.

[0168] Stringent conditions are those that (1) employ low ionic strengthand high temperature for washing, for example, 0.015M NaCl/0.0015Msodium nitrate/0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.

[0169] Another example is use of 50% formamide, 5×SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS. A skilled artisan can readily determine andvary the stringency conditions appropriately to obtain a clear anddetectable hybridization signal.

[0170] Recombinant DNA Molecules Containing PSCA-Encoding Nucleic Acids

[0171] Also provided are recombinant DNA molecules (rDNAs) that containa PSCA-encoding sequences as herein described, or a fragment thereof. Asused herein, a rDNA molecule is a DNA molecule that has been subjectedto molecular manipulation in vitro. Methods for generating rDNAmolecules are well known in the art, for example, see Sambrook et al.,Molecular Cloning (1989). In the preferred rDNA molecules of the presentinvention, a PSCA-encoding DNA sequence that encodes a PSCA protein or afragment of PSCA, is operably linked to one or more expression controlsequences and/or vector sequences. The rDNA molecule can encode eitherthe entire PSCA protein, or can encode a fragment of the PSCA protein.

[0172] The choice of vector and/or expression control sequences to whichthe PSCA-encoding sequence is operably linked depends directly, as iswell known in the art, on the functional properties desired, e.g.,protein expression, and the host cell to be transformed. A vectorcontemplated by the present invention is at least capable of directingthe replication or insertion into the host chromosome, and preferablyalso expression, of the PSCA-encoding sequence included in the rDNAmolecule.

[0173] Expression control elements that are used for regulating theexpression of an operably linked protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, enhancers, transcriptionterminators and other regulatory elements. Preferably, an induciblepromoter that is readily controlled, such as being responsive to anutrient in the host cell's medium, is used.

[0174] In one embodiment, the vector containing a PSCA-encoding nucleicacid molecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule intrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

[0175] Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the PSCA-encoding sequence in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding 6f RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Various viral vectors well known to thoseskilled in the art may also be used, such as, for example, a number ofwell known retroviral vectors.

[0176] Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can also be used to variant rDNAmolecules that contain a PSCA-encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (international Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

[0177] Eukaryotic cell expression vectors used to construct the rDNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. Southern et al., J Mol Anal Genet (1982)1:327-341. Alternatively, the selectable marker can be present on aseparate plasmid, and the two vectors are introduced by cotransfectionof the host cell, and selected by culturing in the presence of theappropriate drug for the selectable marker.

[0178] In accordance with the practice of the invention, the vector canbe a plasmid, cosmid or phage vector encoding the cDNA moleculediscussed above. Additionally, the invention provides a host-vectorsystem comprising the plasmid, cosmid or phage vector transfected into asuitable eukaryotic host cell. Examples of suitable eukaryotic hostcells include a yeast cell, a plant cell, or an animal cell, such as amammalian cell. Examples of suitable cells include the LnCaP, LAPC-4,and other prostate cancer cell lines. The host-vector system is usefulfor the production of a PSCA protein. Alternatively, the host cell canbe prokaryotic, such as a bacterial cell.

[0179] Transformed Host Cells

[0180] The invention further provides host cells transformed with anucleic acid molecule that encodes a PSCA protein or a fragment thereofThe host cell can be either prokaryotic or eukaryotic. Eukaryotic cellsuseful for expression of a PSCA protein are pot limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of a PSCA gene.Preferred eukaryotic host cells include, but are not limited to, yeast,insect and mammalian cells, preferably vertebrate cells such as thosefrom a mouse, rat, monkey or human fibroblastic cell line. Prostatecancer cell lines, such as the LnCaP and LAPC-4 cell lines may also beused. Any prokaryotic host can be used to express a PSCA-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

[0181] Transformation of appropriate cell hosts with an rDNA molecule ofthe present invention is accomplished by well known methods thattypically depend on the type of vector used and host system employed.With regard to transformation of prokaryotic host cells, electroporationand salt treatment methods are typically employed, see, for example,Cohen et al., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with vectors containing rDNAs, electroporation,cationic lipid or salt treatment methods are typically employed, see,for example, Graham et al., Virol (1973) 52:456; Wigler et al., ProcNatl Acad Sci USA (1979) 76:1373-76.

[0182] Successfully transformed cells, i.e., cells that contain an rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

[0183] Recombinant Methods of Generating PSCA Proteins

[0184] The invention further provides methods for producing a PSCAprotein using one of the PSCA-encoding nucleic acid molecules hereindescribed. In general terms, the production of a recombinant PSCAprotein typically can involve the following steps (Maniatis, supra).

[0185] First, a nucleic acid molecule is obtained that encodes a PSCAprotein or a fragment thereof, such as the nucleic acid moleculedepicted in FIG. 1A. The PSCA-encoding nucleic acid molecule is thenpreferably placed in an operable linkage with suitable controlsequences, as described above, to generate an expression unit containingthe PSCA-encoding sequence. The expression unit is used to transform asuitable host and the transformed host is cultured under conditions thatallow the production of the PSCA protein. Optionally the PSCA protein isisolated from the medium or from the cells; recovery and purification ofthe protein may not be necessary in some instances where some impuritiesmay be tolerated.

[0186] Each of the foregoing steps may be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in an appropriate host. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using an appropriate combination of replicons and controlsequences. The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the geneand were discussed in detail earlier. Suitable restriction sites can, ifnot normally available, be added to the ends of the coding sequence soas to provide an excisable gene to insert into these vectors. A skilledartisan can readily adapt any host/expression system known in the artfor use with PSCA-encoding sequences to produce a PSCA protein.

[0187] In a specific embodiment described in the examples which follow,a secreted form of PSCA may be conveniently expressed in 293T cellstransfected with a CMV-driven expression vector encoding PSCA with aC-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen). The secretedHIS-tagged PSCA in the culture media may be purified using a nickelcolumn using standard techniques.

[0188] Assays for Identifying PSCA Ligands and Other Binding Agents

[0189] Another aspect of the invention relates to assays and methodsthat can be used to detect and identify PSCA ligands and other agentsand cellular constituents that bind to PSCA. Specifically, PSCA ligandsand other agents and cellular constituents that bind to PSCA can beidentified by the ability of the PSCA ligand or other agent orconstituent to bind to PSCA and/or the ability to inhibit/stimulate PSCAactivity. Assays for PSCA activity (e.g., binding) using a PSCA proteinare suitable for use in high through-put screening methods.

[0190] In one embodiment, the assay comprises mixing PSCA with a testagent or cellular extract. After mixing under conditions that allowassociation of PSCA with the agent or component of the extract, themixture is analyzed to determine if the agent/component is bound toPSCA. Binding agents/components are identified as being able to bind toPSCA. Alternatively or consecutively, PSCA activity can be directlyassessed as a means for identifying agonists and antagonists of PSCAactivity.

[0191] Alternatively, targets that bind to a PSCA protein can beidentified using a yeast two-hybrid system (Fields, S. and Song, O.(1989), Nature 340:245-246) or using a binding-capture assay (Harlow,supra). In the yeast two hybrid system, an expression unit encoding afusion protein made up of one subunit of a two subunit transcriptionfactor and the PSCA protein is introduced and expressed in a yeast cell.The cell is further modified to contain (1) an expression unit encodinga detectable marker whose expression requires the two subunittranscription factor for expression and (2) an expression unit thatencodes a fusion protein made up of the second subunit of thetranscription factor and a cloned segment of DNA. If the cloned segmentof DNA encodes a protein that binds to the PSCA protein, the expressionresults in the interaction of the PSCA and the encoded protein. Thisbrings the two subunits of the transcription factor into bindingproximity, allowing reconstitution of the transcription factor. Thisresults in the expression of the detectable marker. The yeast two hybridsystem is particularly useful in screening a library of cDNA encodingsegments for cellular binding partners of PSCA.

[0192] PSCA proteins which may be used in the above assays include, butare not limited to, an isolated PSCA protein, a fragment of a PSCAprotein, a cell that has been altered to express a PSCA protein, or afraction of a cell that has been altered to express a PSCA protein.Further, the PSCA protein can be the entire PSCA protein or a definedfragment of the PSCA protein. It will be apparent to one of ordinaryskill in the art that so long as the PSCA protein can be assayed foragent binding, e.g., by a shift in molecular weight or activity, thepresent assay can be used.

[0193] The method used to identify whether an agent/cellular componentbinds to a PSCA protein will be based primarily on the nature of thePSCA protein used. For example, a gel retardation assay can be used todetermine whether an agent binds to PSCA or a fragment thereof.Alternatively, immunodetection and biochip technologies can be adoptedfor use with the PSCA protein. A skilled artisan can readily employnumerous art-known techniques for determining whether a particular agentbinds to a PSCA protein.

[0194] Agents and cellular components can be further tested for theability to modulate the activity of a PSCA protein using a cell-freeassay system or a cellular assay system. As the activities of the PSCAprotein become more defined, functional assays based on the identifiedactivity can be employed.

[0195] As used herein, an agent is said to antagonize PSCA activity whenthe agent reduces PSCA activity. The preferred antagonist willselectively antagonize PSCA, not affecting any other cellular proteins.Further, the preferred antagonist will reduce PSCA activity by more than50%, more preferably by more than 90%, most preferably eliminating allPSCA activity.

[0196] As used herein, an agent is said to agonize PSCA activity whenthe agent increases PSCA activity. The preferred agonist willselectively agonize PSCA, not affecting any other cellular proteins.Further, the preferred antagonist will increase PSCA activity by morethan 50%, more preferably by more than 90%, most preferably more thandoubling PSCA activity.

[0197] Agents that are assayed in the above method can be randomlyselected or rationally selected or designed. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences of the PSCA protein. An example ofrandomly selected agents is the use of a chemical library or a peptidecombinatorial library, or a growth broth of an organism or plantextract.

[0198] As used herein, an agent is said to be rationally selected ordesigned when the agent is chosen on a nonrandom basis that takes intoaccount the sequence of the target site and/or its conformation inconnection with the agent's action. Agents can be rationally selected orrationally designed by utilizing the peptide sequences that make up thePSCA protein. For example, a rationally selected peptide agent can be apeptide whose amino acid sequence is identical to a fragment of a PSCAprotein.

[0199] The agents tested in the methods of the present invention can be,as examples, peptides, small molecules, and vitamin derivatives, as wellas carbohydrates. A skilled artisan can readily recognize that there isno limit as to the structural nature of the agents used in the presentscreening method. One class of agents of the present invention arepeptide agents whose amino acid sequences are chosen based on the aminoacid sequence of the PSCA protein. Small peptide agents can serve ascompetitive inhibitors of PSCA protein assembly.

[0200] Peptide agents can be prepared using standard solid phase (orsolution phase) peptide synthesis methods, as is known in the art. Inaddition, the DNA encoding these peptides may be synthesized usingcommercially available oligonucleotide synthesis instrumentation andproduced recombinantly using standard recombinant production systems.The production using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

[0201] Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the PSCA protein. As describedabove, antibodies are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the PSCA protein intended to be targeted by the antibodies. Criticalregions may include the domains identified in FIG. 15. Such agents, canbe used in competitive binding studies to identify second generationinhibitory agents.

[0202] The cellular extracts tested in the methods of the presentinvention can be, as examples, aqueous extracts of cells or tissues,organic extracts of cells or tissues or partially purified cellularfractions. A skilled artisan can readily recognize that there is nolimit as to the source of the cellular extract used in the screeningmethod of the present invention.

[0203] Agents that bind a PSCA protein, such as a PSCA antibody, can beused to modulate the activity of PSCA, to target anticancer agents toappropriate mammalian cells, or to identify agents that block theinteraction with PSCA. Cells expressing PSCA can be targeted oridentified by using an agent that binds to PSCA.

[0204] How the PSCA binding agents will be used depends on the nature ofthe PSCA binding agent. For example, a PSCA binding agent can be usedto: deliver conjugated toxins, such a diphtheria toxin, cholera toxin,ricin or pseudomonas exotoxin, to a PSCA expressing cell; modulate PSCAactivity; to directly kill PSCA expressing cells; or in screens toidentify competitive binding agents. For example, a PSCA inhibitoryagent can be used to directly inhibit the growth of PSCA expressingcells whereas a PSCA binding agent can be used as a diagnostic agent.

[0205] There are multiple diagnostic uses of the invention. For example,the invention provides methods for diagnosing in a subject, e.g., ananimal or human subject, a cancer associated with the presence of thePSCA protein. In one embodiment, the method comprises quantitativelydetermining the number of PSCA protein in the sample (e.g., cell orbiological fluid sample) using any one or combination of the antibodiesof the invention. Then the number so determined can be compared with theamount in a sample from a normal subject. The presence of a measurablydifferent amount (i.e., the number of PSCA in the test sample exceedsthe number from a normal sample) in the samples indicating the presenceof the cancer. PSCA is overexpressed on a cell when the number of PSCAin the test sample exceeds the number from a normal sample.

[0206] In another embodiment, diagnosis involves quantitativelydetermining in a sample from the subject the amount of RNA encoding thePSCA protein using the nucleic acid of the invention. The amount sodetermined can be compared with the amount of RNA in a sample from anormal subject. Once again, the presence of a measurable differentamount indicating the presence of the cancer.

[0207] Additionally, the invention provides methods for monitoring thecourse of cancer (e.g., prostate, bone metastases of prostate cancer,bladder, pancreatic cancer) or disorders associated with PSCA in asubject by measuring the amount of PSCA in a sample from the subject atvarious points in time. This is done for purposes of determining achange in the amount of PSCA in the sample e.g., to determine whetherthe change is a small change, in the amount or a large change, i.e.,overexpression of PSCA. In one embodiment, the method comprisesquantitatively determining in a first sample from the subject thepresence of a PSCA protein and comparing the amount so determined withthe amount present in a second sample from the subject, such samplesbeing taken at different points in time, a difference in the amountsdetermined being indicative of the course of the cancer.

[0208] In another embodiment, monitoring is effected by quantitativelydetermining in a first sample from the subject the presence of a PSCARNA and comparing the amount so determined with the amount present in asecond sample from the subject, such samples being taken at differentpoints in time, a difference in the amounts determined being indicativeof the course of the cancer (e.g, prostate, bone metastases of prostatecancer, bladder and pancreatic cancer).

[0209] As a further embodiment, the diseases or disorders associatedwith PSCA can be monitored in a sample by detecting an increase in orincreased PSCA gene copy number. An increase in or increased PSCA genecopy number is important because it may correlate with poor outcome.

[0210] The sample can be from an animal or a human. Further, the samplecan be a cell sample. For example, using the methods of the invention,organ tissues such as prostate tissue, bladder tissue, pancreatictissue, neuroendocrine tissue, and bone (any tissue where carcinomas canmetastasize, e.g., node, lung, liver, pancreas) can be evaluated for thepresence of cancer or metastatic lesion. Alternatively, the sample canbe a biological fluid, e.g., urine, blood sera or plasma.

[0211] In accordance with the practice of the invention, detection canbe effected by immunologic detection means involving histology,blotting, ELISA, and ELIFA. When the sample is a tissue or cell sampleit can be formalin-fixed, paraffin-embedded or frozen.

[0212] The invention additionally provides methods of determining adifference in the amount and distribution of PSCA in tissue sectionsfrom a neoplastic tissue to be tested relative to the amount anddistribution of PSCA in tissue sections from a normal tissue. In oneembodiment, the method comprises contacting both the tissue to be testedand the normal tissue with a monoclonal antibody that specifically formsa complex with PSCA and thereby detecting the difference in the amountand distribution of PSCA.

[0213] Further, the invention provides a method for diagnosing aneoplastic or preneoplastic condition in a subject. This methodcomprises obtaining from the subject a sample of a tissue, detecting adifference in the amount and distribution of PSCA in the using themethod above, a distinct measurable difference being indicative of suchneoplastic or preneoplastic condition.

[0214] In accordance with the practice of the invention, the antibodycan be directed to the epitope to which any of the monoclonal antibodiesof the invention is directed. Further, the tissue section can be fromthe bladder, prostate, bone, lymphatic tissues, pancreas, other organs,or muscle.

[0215] The invention also provides methods of detecting andquantitatively determining the concentration of PSCA in a biologicalfluid sample. In one embodiment the method comprises contacting a solidsupport with an excess of one or more monoclonal antibodies which forms(preferably specifically forms) a complex with PSCA under conditionspermitting the monoclonal antibody to attach to the surface of the solidsupport. The resulting solid support to which the monoclonal antibody isattached is then contacted with a biological fluid sample so that thePSCA in the biological fluid binds to the antibody and forms aPSCA-antibody complex. The complexed can be labeled directly orindirectly with a detectable marker. Alternatively, either the PSCA orthe antibody can be labeled before the formation the complex. Thecomplex can then be detected and quantitatively determined therebydetecting and quantitatively determining the concentration of PSCA inthe biological fluid sample. A high concentration of PSCA in the samplerelative to normal cells being indicative of a neoplastic orpreneoplastic condition.

[0216] In accordance with the practice of the invention, the biologicalfluid includes but is not limited to tissue extract, urine, blood,serum, and phlegm. Further, the detectable marker includes but is notlimited to an enzyme, biotin, a fluorophore, a chromophore, a heavymetal, a paramagnetic isotope, or a radioisotope.

[0217] Further, the invention provides a diagnostic kit comprising anantibody that recognizes and binds PSCA (an anti-PSCA antibody); and aconjugate of a detectable label and a specific binding partner of theanti-PSCA antibody. In accordance with the practice of the invention thelabel includes, but is not limited to, enzymes, radiolabels,chromophores and fluorescers.

[0218] Cancer Immunotherapy

[0219] Since PSCA protein is expressed or overexpressed in many cancers,including but not limited to prostate tumors, metastases of prostatetumors (such as bone metastases), bladder cancer and pancreatic cancer,it is a target for cancer immunotherapy. These immunotherapeutic methodsinclude the use of antibody therapy, in vivo vaccines, and ex vivoimmunotherapy approaches.

[0220] In one approach, the invention provides PSCA antibodies that maybe used systemically to treat cancer, such as prostate, bladder andpancreatic cancer. PSCA antibodies may also be useful in the treatmentof various other benign and malignant tumors. Antibodies which bindspecifically to the extracellular domain of PSCA are preferred.Antibodies which target the tumor cells but not the surroundingnon-tumor cells and tissue are preferred. Thus, the invention provides amethod of treating a patient susceptible to or having a cancer whichexpresses PSCA antigen, comprising administering to said patient aneffective amount of an antibody which binds specifically to theextracellular domain of PSCA. In another approach, the inventionprovides a method of inhibiting the growth of tumor cells expressingPSCA, comprising administering to a patient an antibody which bindsspecifically to the extracellular domain of PSCA in an amount effectiveto inhibit growth of the tumor cells. PSCA mAbs may also be used in amethod for selectively inhibiting the growth of or killing a cellexpressing PSCA antigen comprising reacting a PSCA antibodyimmunoconjugate or immunotoxin with the cell in an amount sufficient toinhibit the growth of or kill the cell.

[0221] For example, unconjugated PSCA antibody (including monoclonal,polyclonal, chimeric, humanized, fully human and fragments thereof(e.g., recombinant proteins)) may be introduced into a patient such thatthe antibody binds to PSCA on cancer cells and mediates growthinhibition of such cells (including the destruction thereof), and thetumor, by mechanisms which may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, altering the physiologicfunction of PSCA, and/or the inhibition of ligand binding or signaltransduction pathways. In addition to unconjugated PSCA antibodies,fragments thereof, and recombinant proteins of the invention, PSCAantibodies conjugated to toxic agents such as ricin may also be usedtherapeutically to deliver the toxic agent directly to PSCA-bearingtumor cells and thereby destroy the tumor.

[0222] Cancer immunotherapy using PSCA antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit Rev Inmunol 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186;Tsunenati et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyket al., 1992, Cancer Res 52: 2771-2776), B-cell lymphoma (Funakoshi etal., 1996, J Immunther Emphasis Tumor Immunol 19: 93-101), leukemia(Zhong et al., 1996, Leuk Res 20: 581-589), colorectal cancer (Moun etal., 1994, Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res55: 4398-4403), and breast cancer (Shepard et al., 1991,J Clin Immunol11: 117-127).

[0223] For example, one way to apply antitumor monoclonal antibodiesclinically is to administer them in unmodified form, using monoclonalantibodies of the invention which display antitumor activity (e.g., ADCCand CDC activity) and/or internalizing ability in vitro and/or in animalmodels (see, e.g. Hellstrom et al., Proc. Natl. Acad. Sci. USA82:1499-1502 (1985). To detect ADCC and CDC activity monoclonalantibodies can be tested for lysing cultured ⁵¹Cr-labeled tumor targetcells over a 4-hour incubation period. Target cells are labeled with⁵¹Cr and then can be exposed for 4 hours to a combination of effectorcells (in the form of human lymphocytes purified by the use of alymphocyte-separation medium) and antibody, which is added inconcentrations, e.g., varying between 0.1 μg/ml and 10 μg/ml. Therelease of ⁵¹Cr from the target cells is measured as evidence oftumor-cell lysis (cytotoxicity). Controls include the incubation oftarget cells alone or with either lymphocytes or monoclonal antibodyseparately. The total amount of ⁵¹Cr that can be released is measuredand ADCC is calculated as the percent killing of target cells observedwith monoclonal antibody plus effector cells as compared to target cellsbeing incubated alone. The procedure for CDC is identical to the oneused to detect ADCC except that human serum, as a source of complement,(diluted 1:3 to 1:6) is added in place of the effector cells.

[0224] In the practice of the method of the invention, anti-PSCAantibodies capable of inhibiting the growth of cancer cells expressingPSCA on the cell surface are administered in a therapeutically effectiveamount to cancer patients whose tumors express or overexpress PSCA. Theanti-PSCA mAb therapy method of the invention demonstrates remarkabletumor growth inhibition of prostate tumors in vivo. Accordingly, theinvention provides a specific, effective and long-needed treatment forprostate cancer. The method of the invention may also be useful for thetreatment of other cancers which express or overexpress PSCA, includingbut not limited to bladder carcinoma and pancreatic carcinomas, sinceboth of these cancers express elevated levels of PSCA. The antibodytherapy methods of the invention may be combined with achemotherapeutic, radiation, and/or other therapeutic regimen.

[0225] As described in Example 18A below, individual mouse anti-PSCAmAbs, as well as combinations of these anti-PSCA monoclonal antibodies,are capable of significantly inhibiting prostate tumor growth in vivousing a xenogenic prostate cancer SCID mouse model. In one study, acohort of SCID mice receiving injections of a human prostate tumorxenograft were treated with a combination of several murine anti-PSCAmAbs. The results of this study showed that the treatment was able tocompletely block the formation of tumors in all of these mice—even after61 days post tumor injection. In contrast, all animals in a controlgroup of SCID mice receiving the same prostate tumor xenograft, buttreated with control murine IgG, developed significant and progressivelymore massive tumors during the study. There was no apparent toxicityassociated with the treatment of these animals with the anti-PSCA mAbpreparation, as all mice in the treatment group remained lively andhealthy throughout the experiment. The xenograft used in the study,LAPC-9, was generated from a bone tumor biopsy of a patient withhormone-refractory metastatic prostate cancer, is characterized by anextremely androgen-sensitive phenotype (PSA levels drop to zero aftercastration in recipient SCID mice), particularly aggressive growthproperties, and high level overexpression of PSCA. LAPC-9 and isdescribed further elsewhere (Published PCT Application WO98/16628,Sawyers et al., Apr. 23, 1998). These results were confirmed in a secondin vivo study described in Example 18B. In addition, further in vivostudies demonstrated that anti-PSCA mAbs are therapeutically effectivewhen used alone (Example 18C1, C2). In all of these in vivo studies,tumors in mice receiving the anti-PSCA mAb treatments had significantlyslower growth rates, longer latency periods, and were smaller in sizecompared to tumors in mice receiving control antibody treatments. SerumPSA levels were also lower in relation to control treated animals andcorrelated with tumor inhibition. Moreover, antibodies recognizingdifferent PSCA epitopes, as well as antibodies having different IgGisotypes, are, therapeutically effective. In one study, anti-PSCA mAbseffectively inhibited the growth of established prostate tumors in vivo(Example 18, C4). Some of the mice treated in this particular studyshowed tumor regression following PSCA treatment (Example 18).

[0226] Additionally, the 3C5 antibody, administered to a tumor-bearingmouse, targeted the tumor cells that express PSCA. A SCID mouse bearingan LAPC-9 tumor (e.g., expressed PSCA), was treated with 3C5 antibody.The tumor was explanted and examined for the presence of the 3C5antibody, by immunohistochemistry analysis (Example 26, FIG. 71). Thefixed tissue slices were probed with goat anti-mouse IgG. The 3C5antibody was localized to the mass of PSCA-expressing tumor cells (FIG.71) and could be detected throughout the tumor. Because SCID miceproduce no immunoglobulin, the antibody detected in the tumor tissuemost likely originated from the 3C5 treatment. To confirm thelocalization of the 3C5 antibody, Western blot analysis was performed ontumor explants from the same mouse. The blot included protein extractsfrom the tumor explant, control IgG antibody, and 3C5 antibody, and theblot was probed with goat anti-mouse IgG-HRP antibodies. The IgG heavyand light chains were readily detected in the tumor lysates from the3C5-treated mouse (FIG. 72).

[0227] The results of a different study also indicate that anti-PSCAantibodies can target PSCA-expressing tumors. A SCID mouse bearing anestablished LAPC-9 tumor was treated with 1G8 antibody. The explantedtumor was examined for the presence of the 1G8 antibody, by Western blotanalysis (Example 26, FIG. 72) using goat anti-mouse IgG-HRP antibodiesas a probe. The heavy and light chains were readily detectable in the1G8-treated mouse. These results indicate that anti-PSCA antibodiesadministered to an subject, can circulate and target a PSCA-expressingtumor. This suggests that anti-PSCA monoclonal antibodies can circulateand target PSCA-expressing cells in tumors that are local, locallyrecurring, and metastatic. Furthermore, this suggests that conjugatedanti-PSCA monoclonal antibodies can target and kill tumors cellsexpressing PSCA.

[0228] As described in Example 24 below, individual anti-PSCA mAbs arecapable of inhibiting prostate tumor growth in vivo, in a xenogenicprostate cancer SCID mouse model. For example, two cohorts of SCID micereceived injections of LAPC-9, and were treated with 1G8 or 3C5. Theresults showed that treatment with 1G8 or 3C5 alone inhibited tumorgrowth in the tumor-bearing mice. In contrast, the mice in a controlgroup that received the same prostate tumor xenograft, but treated withmurine IgG or phosphate buffer, developed larger tumors during thestudy. In addition, the anti-PSCA treatment significantly prolonged thelife of the mice receiving the antibody treatment, compared to thecontrol mice. The prolonged life of the antibody-treated mice correlatedwith a decrease in tumor growth, and effected the level of serum PSAlevels. These results indicate that treatment with anti-PSCA antibodycan prolong the life of a tumor-bearing animal, by inhibiting tumorgrowth.

[0229] The effect of anti-PSCA mAbs in combination with an cytotoxicagent was also tested. As described in Example 25 below, two cohorts ofSCID mice received injections of PC3 cells which were engineered toexpress PSCA, and the mice were treated with 1G8 alone or in combinationwith doxorubicin. The results showed that treatment with 1G8 inhibitedtumor growth of the PSCA-positive PC3 cells, and the combination of 1G8and doxorubicin had a synergetic effect on inhibiting tumor growth,compared to the tumors in mice treated with phosphate buffer ordoxorubicin alone.

[0230] Thus, the results of Example 24 show that anti-PSCA monoclonalantibodies, having different isotypes, are effective in inhibiting thegrowth of established androgen-dependent tumors. For example, the LAPC-9xenograft was generated from a bone tumor biopsy of a patient withhormone-refractory metastatic prostate cancer. The 1G8 antibody is amouse gamma-1, isotypic, neutral antibody, which interacts directly withthe PSCA antigen. The 3C5 antibody is a mouse gamma-2A isotypic,antibody, which binds to cells and complement. Thus, the 1G8 antibodymay direct cell cytotoxicity of androgen-dependent tumors, through anantibody-dependent cell cytotoxicity (ADCC) mechanism, and the 3C5antibody may initiate a potent immune response against the tumor. In asimilar manner, the results of Example 25 show that anti-PSCA antibodiesare effective in treating established androgen-independent tumors.

[0231] Patients may be evaluated for the presence and level of PSCAoverexpression in tumors, preferably using immunohistochemicalassessments of tumor tissue, quantitative PSCA imaging, or othertechniques capable of reliably indicating the presence and degree ofPSCA expression. Immunohistochemical analysis of tumor biopsies orsurgical specimens may be preferred for this purpose. Methods forimmunohistochemical analysis of tumor tissues are well known in the art.An example of an immunohistochemical analytical technique useful fordetermining the level of PSCA overexpression in a sample is described inthe example sections below.

[0232] Anti-PSCA monoclonal antibodies useful in treating cancer includethose which are capable of initiating a potent immune response againstthe tumor and those which are capable of direct cytotoxicity. In thisregard, anti-PSCA mAbs may elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites or complement proteins. In addition, anti-PSCA mAbs which exert adirect biological effect on tumor growth are useful in the practice ofthe invention. Such mAbs may not require the complete immunoglobulin toexert the effect. Potential mechanisms by which such directly cytotoxicmAbs may act include inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism by which a particularanti-PSCA mAb exerts an anti-tumor effect may be evaluated using anynumber of in vitro assays designed to determine ADCC, ADMMC,complement-mediated cell lysis, and so forth, such as those described inExample 19, below.

[0233] The anti-tumor activity of a particular anti-PSCA mAb, orcombination of anti-PSCA mAbs, is preferably evaluated in vivo using asuitable animal model. Xenogenic cancer models, wherein human cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice, are particularlyappropriate and are known. Examples of xenograft models of humanprostate cancer (capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease) are described in Klein et al.,1997, Nature Medicine 3: 402-408 and in PCT Patent ApplicationWO98/16628, Sawyers et al., published Apr. 23, 1998. The examples hereinprovide detailed experimental protocols for evaluating the anti-tumorpotential of anti-PSCA mAb preparations in vivo. Other in vivo assaysare contemplated, such as those which measure regression of establishedtumors, interference with the development of metastasis, and the like.

[0234] It should be noted that the use of murine or other non-humanmonoclonal antibodies and chimeric mAbs may induce moderate to strongimmune responses in some patients. In the most severe cases, such animmune response may lead to the extensive formation of immune complexeswhich, potentially, can cause renal failure. Accordingly, preferredmonoclonal antibodies used in the practice of the therapeutic methods ofthe invention are those which are either fully human or humanized andwhich bind specifically to the target PSCA antigen with high affinitybut exhibit low or no antigenicity in the patient.

[0235] The method of the invention contemplate the administration ofsingle anti-PSCA mAbs as well as combinations, or “cocktails, ofdifferent individual mAbs such as those recognizing different epitopes.Such mAb cocktails may have certain advantages inasmuch as they containmAbs which bind to different epitopes and/or exploit different effectormechanisms or combine directly cytotoxic mAbs with mAbs that rely onimmune effector functionality. Such mAbs in combination may exhibitsynergistic therapeutic effects. In addition, the administration ofanti-PSCA mAbs may be combined with other therapeutic agents, includingbut not limited to various chemotherapeutic agents, androgen-blockers,and immune modulators (e.g., IL-2, GM-CSF). The anti-PSCA mAbs may beadministered in their “naked” or unconjugated form, or may havetherapeutic agents conjugated to them.

[0236] The anti-PSCA monoclonal antibodies used in the practice of themethod of the invention may be formulated into pharmaceuticalcompositions comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which when combined withthe anti-PSCA mAbs retains the anti-tumor function of the antibody andis non-reactive with the subject's immune systems. Examples include, butare not limited to, any of a number of standard pharmaceutical carrierssuch as sterile phosphate buffered saline solutions, bacteriostaticwater, and the like (see, generally, Remington's Pharmaceutical Sciences16^(th) Edition, A. Osal., Ed., 1980).

[0237] The anti-PSCA antibody formulations may be administered via anyroute capable of delivering the antibodies to the tumor site.Potentially effective routes of administration include, but are notlimited to, intravenous, intraperitoneal, intramuscular, intratumor,intradermal, and the like. The preferred route of administration is byintravenous injection. A preferred formulation for intravenous injectioncomprises the anti-PSCA mAbs in a solution of preserved bacteriostaticwater, sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. The anti-PSCA mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

[0238] Treatment will generally involve the repeated administration ofthe anti-PSCA antibody preparation via an acceptable route ofadministration such as intravenous injection (IV), at an effective dose.Dosages will depend upon various factors generally appreciated by thoseof skill in the art, including without limitation the type of cancer andthe severity, grade, or stage of the cancer, the binding affinity andhalf life of the mAb or mAbs used, the degree of PSCA expression in thepatient, the extent of circulating shed PSCA antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic agents used in combination with thetreatment method of the invention. Typical daily doses may range fromabout 0.1 to 100 mg/kg. Doses in the range of 10-500 mg mAb per week maybe effective and well tolerated, although even higher weekly doses maybe appropriate and/or well tolerated. The principal determining factorin defining the appropriate dose is the amount of a particular antibodynecessary to be therapeutically effective in a particular context.Repeated administrations may be required in order to achieve tumorinhibition or regression. Initial loading doses may be higher. Theinitial loading dose may be administered as an infusion. Periodicmaintenance doses may be administered similarly, provided the initialdose is well tolerated.

[0239] Direct administration of PSCA mAbs is also possible and may haveadvantages in certain contexts. For example, for the treatment ofbladder carcinoma, PSCA mAbs may be injected directly into the bladder.Because PSCA mAbs administered directly to bladder will be cleared fromthe patient rapidly, it may be possible to use non-human or chimericantibodies effectively without significant complications ofantigenicity.

[0240] Patients may be evaluated for serum PSCA in order to assist inthe determination of the most effective dosing regimen and relatedfactors. The PSCA Capture ELISA described in Example 20 infra, or asimilar assay, may be used for quantitating circulating PSCA levels inpatients prior to treatment. Such assays may also be used for monitoringpurposes throughout therapy, and may be useful to gauge therapeuticsuccess in combination with evaluating other parameters such as serumPSA levels.

[0241] The invention further provides vaccines formulated to contain aPSCA protein or fragment thereof. The use of a tumor antigen in avaccine for generating humoral and cell-mediated immunity for use inanti-cancer therapy is well known in the art and, for example, has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117). Such methods can be readily practiced byemploying a PSCA protein, or fragment thereof, or a PSCA-encodingnucleic acid molecule and recombinant vectors capable of expressing andappropriately presenting the PSCA immunogen.

[0242] For example, viral gene delivery systems may be used to deliver aPSCA-encoding nucleic acid molecule. Various viral gene delivery systemswhich can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a PSCA protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human PSCA cDNA may be employed. In anotherembodiment, PSCA nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a PSCA protein which are capable of optimallybinding to specified HLA alleles.

[0243] Various ex vivo strategies may also be employed. One approachinvolves the use of dendritic cells to present PSCA antigen to apatient's immune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to present PSCApeptides to T cells in the context of MHC class I and II molecules. Inone embodiment, autologous dendritic cells are pulsed with PSCA peptidescapable of binding to MHC molecules. In another embodiment, dendriticcells are pulsed with the complete PSCA protein. Yet another embodimentinvolves engineering the overexpression of the PSCA gene in dendriticcells using various implementing vectors known in the art, such asadenovirus (Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus(Henderson et al., 1996, Cancer Res. 56: 3763-3770), lentivirus,adeno-associated virus, DNA transfection (Ribas et al., 1997, CancerRes. 57: 2865-2869), and tumor-derived RNA transfection (Ashley et al.,1997, J. Exp. Med. 186: 1177-1182).

[0244] Anti-idiotypic anti-PSCA antibodies can also be used inanti-cancer therapy as a vaccine for inducing an immune response tocells expressing a PSCA protein. Specifically, the generation ofanti-idiotypic antibodies is well known in the art and can readily beadapted to generate anti-idiotypic anti-PSCA antibodies that mimic anepitope on a PSCA protein (see, for example, Wagner et al., 1997,Hybridoma 16: 33-40; Foon et al., 1995, J Clin Invest 96: 334-342;Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76)., Such ananti-idiotypic antibody can be used in anti-idiotypic therapy aspresently practiced with other anti-idiotypic antibodies directedagainst tumor antigens.

[0245] Genetic immunization methods may be employed to generateprophylactic or therapeutic humoral and cellular immune responsesdirected against cancer cells expressing PSCA. Using the PSCA-encodingDNA molecules described herein, constructs comprising DNA encoding aPSCA protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded PSCA protein/immunogen. The PSCA protein/immunogen may beexpressed as a cell surface protein or be secreted. Expression of thePSCA protein/immunogen results in the generation of prophylactic ortherapeutic humoral and cellular immunity against prostate cancer.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used (for review, see information and referencespublished at internet address www.genweb.com).

[0246] The invention further provides methods for inhibiting cellularactivity (e.g., cell proliferation, activation, or propagation) of acell expressing multiple PSCA antigens on its cell surface. This methodcomprises reacting the immunoconjugates of the invention (e.g., aheterogeneous or homogenous mixture) with the cell so that the PSCAantigens on the cell surface forms a complex with the immunoconjugates.The greater the number of PSCA antigens on the cell surface, the greaterthe number of PSCA-antibody complexes can be used. The greater thenumber of PSCA-antibody complexes the greater the cellular activity thatis inhibited. A subject with a neoplastic or preneoplastic condition canbe treated in accordance with this method when the inhibition ofcellular activity results in cell death.

[0247] A heterogeneous mixture includes PSCA antibodies that recognizedifferent or the same epitope, each antibody being conjugated to thesame or different therapeutic agent. A homogenous mixture includesantibodies that recognize the same epitope, each antibody beingconjugated to the same therapeutic agent.

[0248] The invention further provides methods for inhibiting thebiological activity of PSCA by blocking PSCA from binding its ligand.The methods comprises contacting an amount of PSCA with an antibody orimmunoconjugate of the invention under conditions that permit aPSCA-immunoconjugate or PSCA-antibody complex thereby blocking PSCA frombinding its ligand and inhibiting the activity of PSCA.

[0249] In another embodiment, the invention provides methods forselectively inhibiting a cell expressing PSCA antigen by reacting anyone or a combination of the immunoconjugates of the invention with thecell in an amount sufficient to inhibit the cell. Such amounts includean amount to kill the cell or an amount sufficient to inhibit cellgrowth or proliferation. As discussed supra the dose and dosage regimenwill depend on the nature of the disease or disorder to be treatedassociated with PSCA, its population, the site to which the antibodiesare to be directed, the characteristics of the particular immunotoxin,and the patient. For example, the amount of immunoconjugate can be inthe range of 0.1 to 200 mg/kg of patient weight.

[0250] Methods for Identifying PSCA Proteins and PSCA Genes and RNA

[0251] The invention provides methods for identifying cells, tissues ororganisms expressing a PSCA protein or a PSCA gene. Such methods can beused to diagnose the presence of cells or an organism that expresses aPSCA protein in vivo or in vitro. The methods of the present inventionare particularly useful in the determining the presence of cells thatmediate pathological conditions of the prostate. Specifically, thepresence of a PSCA protein can be identified by determining whether aPSCA protein, or nucleic acid encoding a PSCA protein, is expressed. Theexpression of a PSCA protein can be used as a means for diagnosing thepresence of cells, tissues or an organism that expresses a PSCA proteinor gene.

[0252] A variety of immunological and molecular genetic techniques canbe used to determine if a PSCA protein is expressed/produced in aparticular cell or sample. In general, an extract containing nucleicacid molecules or an extract containing proteins is prepared. Theextract is then assayed to determine whether a PSCA protein, or aPSCA-encoding nucleic acid molecule, is produced in the cell.

[0253] Various polynucleotide-based detection methods well known in theart may be employed for the detection of PSCA-encoding nucleic acidmolecules and for the detection of PSCA expressing cells in a biologicalspecimen. For example, RT-PCR methods may be used to selectively amplifya PSCA mRNA or fragment thereof, and such methods may be employed toidentify cells expressing PSCA, as described in Example 1 below. In aparticular embodiment, RT-PCR is used to detect micrometastaticprostate, bladder or pancreatic cancer cells or circulating prostate,bladder or pancreatic cancer cells. Various other amplification typedetection methods, such as, for example, branched DNA methods, andvarious well known hybridization assays using DNA or RNA probes may alsobe used for the detection of PSCA-encoding polynucleotides or PSCAexpressing cells.

[0254] Various methods for the detection of proteins are well known inthe art and may be employed for the detection of PSCA proteins and PSCAexpressing cells. To perform a diagnostic test based on proteins, asuitable protein sample is obtained and prepared using conventionaltechniques. Protein samples can be prepared, for example, simply byboiling a sample with SDS. The extracted protein can then be analyzed todetermine the presence of a PSCA protein using known methods. Forexample, the presence of specific sized or charged variants of a proteincan be identified using mobility in an electric filed. Alternatively,antibodies can be used for detection purposes. A skilled artisan canreadily adapt known protein analytical methods to determine if a samplecontains a PSCA protein.

[0255] Alternatively, PSCA expression can also be used in methods toidentify agents that decrease the level of expression of the PSCA gene.For example, cells or tissues expressing a PSCA protein can be contactedwith a test agent to determine the effects of the agent on PSCAexpression. Agents that activate PSCA expression can be used as anagonist of PSCA activity whereas agents that decrease PSCA expressioncan be used as an antagonist of PSCA activity.

[0256] PSCA Promoter and Other Expression Regulatory Elements

[0257] The invention further provides expression control sequences found5′ of the of the newly identified PSCA gene in a form that can be usedto generate expression vectors and transgenic animals. Specifically, thePSCA expression control elements, such as the PSCA promoter that canreadily be identified as being 5′ from the ATG start codon in the PSCAgene, and can be used to direct the expression of an operably linkedprotein encoding DNA sequence. Since PSCA expression is predominantlyexpressed in prostate cells, the expression control elements areparticularly useful in directing the expression of an introducedtransgene in a tissue specific fashion. A skilled artisan can readilyuse the PSCA gene promoter and other regulatory elements in expressionvectors using methods known in the art.

[0258] In eukaryotic cells, the regulatory sequences can be foundupstream, downstream and within the coding region of the gene. Theeukaryotic regulatory sequences comprise a promoter sequence andsometimes at least one enhancer sequence. In a typical eukaryotic gene,the promoter sequence resides upstream and proximal to the coding regionof the gene, and must be oriented in one direction to control expressionof the gene. In a typical eukaryotic gene, the enhancer sequences canreside in the upstream, downstream and even within the coding region ofthe gene, and can be oriented in either direction to enhance or suppressexpression of the gene.

[0259] The present invention provides a DNA fragment containing 9 kb ofsequences upstream of the PSCA coding region. The ability of this PSCAfragment to drive expression of an operatively linked transgene has beentested using a series of chimeric reporter constructs transfected intocells. The chimeric reporter constructs demonstrate an expressionpattern similar to that of native endogenous PSCA, and the PSCA fragmentdrives expression of the transgene when linked in the forwardorientation. Thus, this PSCA fragment comprises a PSCA upstreamregulatory region that exhibits promoter-like activity.

[0260] PSCA transcripts are also present at a significantly higher levelin prostate tumor cells but not in benign prostatic hyperplasia. ThusPSCA transcripts are detectable in a prostate-predominant manner, andare detectable at a higher level in prostate tumor samples. Thesignificantly higher level of PSCA transcripts, or over-expression as isknown in the art, can be determined by measuring and comparing theamount of detectable PSCA transcripts in a normal prostate with aprostate tumor sample. This comparison can be performed by methods wellknown in the art, including Northern analysis and RT-PCR assays, and thedifferences in transcript levels can be quantitated. Thus, the presenceof a measurably different amount of PSCA transcripts (i.e., the numberof PSCA transcripts in the test sample exceeds the number from a normalsample) in the samples can be used to indicate the presence of prostatecancer.

[0261] PSCA expression is also observed in other human cancers,particularly bladder and pancreatic carcinomas. In the case of bladdercarcinoma, the degree of PSCA expression appears to correlate with theseverity of the disease, reaching the highest level of overexpression ininvasive bladder cancer (See Example 17, below).

[0262] The pattern of PSCA transcript and protein accumulation is known,and the PSCA upstream regulatory region has been isolated andcharacterized. A series of chimeric constructs comprising the PSCAupstream regulatory region operatively linked to a transgene has beentested. The PSCA upstream regulatory region drives expression of thetransgene in various prostate cells and cell lines, and in bladder, andto a lesser extent in kidney. Thus, the PSCA upstream region drivesexpression of a transgene in a prostate-predominant manner.

[0263] In preferred embodiments, DNA fragments of 9kb, 6kb, 3kb, and 1kbderived from the 5′ upstream region of the PSCA gene, as shown in FIG.42, were produced by techniques described herein. The 9kb PSCA upstreamregion (pEGFP-PSCA) is involved with gene regulatory activity and wasdeposited on May 17, 1999 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 and has there beenidentified as follows ATCC No. PTA-80. The 9 kb fragment was obtained byamplification using a T7 primer and RIhPSCA3′-5(5′-gggaattcgcacagccttcagggtc-3′).

[0264] Uses of The Fragment Having Gene Regulatory Activity

[0265] This invention provides methods (e.g., gene therapy methods) fortargeting a gene-of-interest to a cancer cell/site so that the proteinencoded by the gene can be expressed thereby directly or indirectlyameliorating the diseased state.

[0266] A susceptible cell is introduced with an expression vector thatexpresses a transgene (e.g., a therapeutic gene) under the control of aPSCA upstream region having significantly increased gene expressionactivity in tumor cells. The use of an expression vector that expressesa therapeutic gene predominantly in tumor cells will allow expression ofthe therapeutic genes in target cell, such as prostate, bladder andpancreatic tumor cells.

[0267] After infecting a susceptible cell, a transgene (e.g., atherapeutic gene) is driven by a PSCA upstream region having increasedgene expression activity in a vector, that expresses the protein encodedby the transgene. The use of a fairly specific prostate specific genevector will allow selective expression of the specific genes in targetcells, e.g., prostate cancer cells.

[0268] PSCA regions having increased gene expression activity may bemodified, e.g., by sequence mutations, deletions, and insertions, so asto produce derivative molecules. Modifications include multiplying thenumber of sequences that can bind prostate cell specific regulatoryproteins and deleting sequences that are nonfunctional in the PSCAregion having gene expression activity. Other modifications includeadding enhancers thereby improving the efficiency of the PSCA regionhaving promoter activity. Enhancers may function in aposition-independent manner and can be located upstream, within ordownstream of the transcribed region.

[0269] Derivative molecules would retain the functional property of thePSCA upstream region having increased gene expression activity, namely,the molecule having such substitutions will still permit substantiallyprostate tissue specific expression of a gene of interest located 3′ tothe fragment. Modification is permitted so long as the derivativemolecules retain its ability to drive gene expression in a substantiallyprostate specific manner compared to a PSCA fragment having promoteractivity alone.

[0270] In a preferred embodiment, a vector was constructed by insertinga heterologous sequence (therapeutic gene) downstream of the PSCAupstream region having promoter activity.

[0271] Examples of therapeutic genes include suicide genes. These aregenes sequences the expression of which produces a protein or agent thatinhibits tumor cell growth or tumor cell death (e.g., prostate tumorcells). Suicide genes include genes encoding enzymes (e.g., prodrugenzymes), oncogenes, tumor suppressor genes, genes encoding toxins,genes encoding cytokines, or a gene encoding oncostatin. The purpose ofthe therapeutic gene is to inhibit the growth of or kill the cancer cellor produce cytokines or other cytotoxic agents which directly orindirectly inhibit the growth of or kill the cancer cells.

[0272] Suitable prodrug enzymes include thymidine kinase (TK),xanthine-guanine phosphoribosyltransferase (GPT) gene from E. Coli or E.Coli cytosine deaminase (CD), or hypoxanthine phosphoribosyl transferase(HPRT).

[0273] Suitable oncogenes and tumor suppressor genes include neu, EGF,ras (including H, K, and N ras), p53, Retinoblastoma tumor suppressorgene (Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase(PTPase), and nm23. Suitable toxins include Pseudomonas exotoxin A andS; diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-liketoxins (SLT-1, -2), ricin, abrin, supporin, and gelonin.

[0274] Suitable cytokines include interferons, GM-CSF interleukins,tumor necrosis factor (TNF) (Wong G, et al., Human GM-CSF: Molecularcloning of the complementary DNA and purification of the natural andrecombinant proteins. Science 1985; 228:810); WO9323034 (1993);Horisberger M A, et al., Cloning and sequence analyses of cDNAs forinterferon-and virus-induced human Mx proteins reveal that they containputative guanine nucleotide-binding sites: functional study of thecorresponding gene promoter. Journal of Virology, March 1990,64(3):1171-81; Li YP et al., Proinflammatory cytokines tumor necrosisfactor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin genepromoter. Journal of Immunology, Feb. 1, 1992 148(3):788-94; Pizarro TT, et al. Induction of TNF alpha and TNF beta gene expression in ratcardiac transplants during allograft rejection. Transplantations August1993, 56(2):399404). (Breviario F, et al., interleukin-l-inducible genesin endothelial cells. Cloning of a new gene related to C-reactiveprotein and serum amyloid P component. Journal of Biological Chemistry,Nov. 5, 1992 267(31):22190-7; Espinoza-Delgado I, et al., Regulation ofIL2 receptor subunit genes in human monocytes. Differential effects ofIL-2 and IFN-gamma. Journal of Immunology, Nov. 1, 1992 149(9):2961-8;Algate P A, et al., Regulation of the interleukin-3 (IL-3) receptor byIL-3 in the fetal liver-derived FL5.12 cell line. Blood, May 1, 199483(9):2459-68; Cluitmans F H, et al., IL4 down-regulates IL,2-, IL-3-,and GM-CSF-induced cytokine gene expression in peripheral bloodmonocytes. Annals of Hematology, June 1994, 68(6):293-8; Lagoo, A S, etal., IL-2, IL4, and IFN-gamma gene expression versus secretion insuperantigen-activated T cells. Distinct requirement for costimulatorysignals through adhesion molecules. Journal of Immunology, Feb. 15, 1994152(4):1641-52; Martinez O M, et al., IL-2 and IL-5 gene expression inresponse to alloantigen in liver allograft recipients and in vitro.Transplantation, May, 1993, 55(5):1159-66; Pang G, et al., GM-CSF, IL-1alpha, IL-1 beta, IL6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expressionand cytokine production in human duodenal fibroblasts stimulated withlipopolysaccharide, IL-1 alpha and TNF-alpha. Clinical and ExperimentalImmunology, June 1994, 96(3):43743; Ulich T R, et al., Endotoxin-inducedcytokine gene expression in vivo. III. IL-6 mRNA and serum proteinexpression and the in vivo hematologic effects of IL-6. Journal ofImmunology, Apr. 1, 1991 146(7):2316-23; Mauviel A, et al.,Leukoregulin, a T cell-derived cytokine, induces IL-8 gene expressionand secretion in human skin fibroblasts. Demonstration and secretion inhuman skin fibroblasts. Demonstration of enhanced NF-kappa B binding andNF-kappa B-driven promoter activity. Journal of Immunology, Nov. 1, 1992149(9):2969-76).

[0275] Growth factors include Transforming Growth Factor-α (TGFα) and β(TGFβ), cytokine colony stimulating factors (Shimane M, et al.,Molecular cloning and characterization of G-CSF induced gene cDNA.Biochemical and Biophysical Research Communications, Feb. 28, 1994199(l):26-32; Kay A B, et al., Messenger RNA expression of the cytokinegene cluster, interleukin 3 (IL-3), IL-4, IL-5, andgranulocyte/macrophage colony-stimulating factor, in allergen-inducedlate-phase cutaneous reactions in atopic subjects. Journal ofExperimental Medicine, Mar. 1, 1991 173(3):775-8; de Wit H, et al.,Differential regulation of M-CSF and IL,6 gene expression in monocyticcells. British Journal of Haematology, February 1994, 86(2):259-64;Sprecher E, et al., Detection of IL-1 beta, TNF-alpha, and IL-6 genetranscription by the polymerase chain reaction in keratinocytes,Langerhans cells and peritoneal exudate cells during infection withherpes simplex virus-1. Archives of Virology, 1992, 126(14):253-69).

[0276] Vectors suitable for use in the methods of the present inventionare viral vectors including adenoviruses, lentivirus, retroviralvectors, adeno-associated viral (AAV) vectors.

[0277] Preferably, the viral vector selected should meet the followingcriteria: 1) the vector must be able to infect the tumor cells and thusviral vectors having an appropriate host range must be selected; 2) thetransferred gene should be capable of persisting and being expressed ina cell for an extended period of time; and 3) the vector should be safeto the host and cause minimal cell transformation. Retroviral vectorsand adenoviruses offer an efficient, useful, and presently thebest-characterized means of introducing and expressing foreign genesefficiently in mammalian cells. These vectors have very broad host andcell type ranges, express genes stably and efficiently. The safety ofthese vectors has been proved by many research groups. In fact many arein clinical trials.

[0278] Other virus vectors that may be used for gene transfer into cellsfor correction of disorders include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma;Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovine papilloma virustype I (BPV); vaccinia and poliovirus and other human and animalviruses.

[0279] Adenoviruses have several properties that make them attractive ascloning vehicles (Bachettis et al.: Transfer of gene for thymidinekinase-deficient human cells by purified herpes simplex viral DNA. PNASUSA, 1977 74:1590; Berkner, K. L.: Development of adenovirus vectors forexpression of heterologous genes. Biotechniques, 1988 6:616;Ghosh-Choudhury G, et al., Human adenovirus cloning vectors based oninfectious bacterial plasmids. Gene 1986; 50:161; Hag-Ahmand Y. et al.,Development of a helper-independent human adenovirus vector and its usein the transfer of the herpes simplex virus thymidine kinase gene. JVirol 1986; 57:257; Rosenfeld M, et al., Adenovirus-mediated transfer ofa recombinant al-antitrypsin gene to the lung epithelium in vivo.Science 1991; 252:431).

[0280] For example, adenoviruses possess an intermediate sized genomethat replicates in cellular nuclei; many serotypes are clinicallyinnocuous; adenovirus genomes appear to be stable despite insertion offoreign genes; foreign genes appear to be maintained without loss orrearrangement; and adenoviruses can be used as high level transientexpression vectors with an expression period up to 4 weeks to severalmonths. Extensive biochemical and genetic studies suggest that it ispossible to substitute up to 7-7.5 kb of heterologous sequences fornative adenovirus sequences generating viable, conditional,helper-independent vectors (Kaufman R. J.; identification of thecomponent necessary for adenovirus translational control and theirutilization in cDNA expression vectors. PNAS USA, 1985 82:689).

[0281] AAV is a small human parvovirus with a single stranded DNA genomeof approximately 5 kb. This virus can be propagated as an integratedprovirus in several human cell types. AAV vectors have several advantagefor human gene therapy. For example, they are trophic for human cellsbut can also infect other mammalian cells; (2) no disease has beenassociated with AAV in humans or other animals; (3) integrated AAVgenomes appear stable in their host cells; (4) there is no evidence thatintegration of AAV alters expression of host genes or promoters orpromotes their rearrangement; (5) introduce genes can be rescued fromthe host cell by infection with a helper virus such as adenovirus.

[0282] HSV-1 vector system facilitates introduction of virtually anygene into non-mitotic cells (Geller et al. an efficient deletion mutantpackaging system for a defective herpes simplex virus vectors: Potentialapplications to human gene therapy and neuronal physiology. PNAS USA,1990 87:8950).

[0283] Another vector for mammalian gene transfer is the bovinepapilloma virus-based vector (Sarver N, et al., Bovine papilloma virusDNA: A novel eukaryotic cloning vector. Mol Cell Biol 1981; 1:486).

[0284] Vaccinia and other poxvirus-based vectors provide a mammaliangene transfer system. Vaccinia virus is a large double-stranded DNAvirus of 120 kilodaltons (kd) genomic size (Panicali D, et al.,Construction of poxvirus as cloning vectors: Insertion of the thymidinekinase gene from herpes simplex virus into the DNA of infectious vaccinevirus. Proc Natl Acad Sci USA 1982; 79:4927; Smith et al. infectiousvaccinia virus recombinants that express hepatitis B virus surfaceantigens. Nature, 1983 302:490.)

[0285] Retroviruses are packages designed to insert viral genes intohost cells (Guild B, et al., Development of retrovirus vectors usefulfor expressing genes in cultured murine embryonic cells andhematopoietic cells in vivo. J Virol 1988; 62:795; Hock R A, et al.,Retrovirs mediated transfer and expression of drug resistance genes inhuman hemopoietic progenitor cells. Nature 1986; 320:275).

[0286] The basic retrovirus consists of two identical strands of RNApackaged in a proviral protein. The core surrounded by a protective coatcalled the envelope, which is derived from the membrane of the previoushost but modified with glycoproteins contributed by the virus.

[0287] Preferably, for treating defects, disease or damage of cells in,for example, the prostate, vectors of the invention include atherapeutic gene or transgenes, for example a gene encoding TK. Thegenetically modified vectors are administered into the prostate to treatdefects, disease such as prostate cancer by introducing a therapeuticgene product or products into the prostate that enhance the productionof endogenous molecules that have ameliorative effects in vivo. The sameprinciples apply with respect to the treatment of other cancers, such aspancreatic, bladder or other cancers expressing PSCA.

[0288] The basic tasks in this embodiment of the present method of theinvention are isolating the gene of interest, attaching it to a fragmenthaving gene regulatory activity, selecting the proper vector vehicle todeliver the gene of interest to the body, administering the vectorhaving the gene of interest into the body, and achieving appropriateexpression of the gene of interest to a target cell. The presentinvention provides packaging the cloned genes, i.e. the genes ofinterest, in such a way that they can be injected directly into thebloodstream or relevant organs of patients who need them. The packagingwill protect the foreign DNA from elimination by the immune system anddirect it to appropriate tissues or cells.

[0289] Along with the human or animal gene of interest another gene,e.g., a selectable marker, can be inserted that will allow easyidentification of cells that have incorporated the modified retrovirus.The critical focus on the process of gene therapy is that the new genemust be expressed in target cells at an appropriate level with asatisfactory duration of expression.

[0290] The methods described below to modify vectors and administeringsuch modified vectors into the target organ (e.g., prostate) are merelyfor purposes of illustration and are typical of those that might beused. However, other procedures may also be employed, as is understoodin the art.

[0291] Most of the techniques used to construct vectors and the like arewidely practiced in the art, and most practitioners are familiar withthe standard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs may serveas a guideline.

[0292] General Methods for Vector Construction

[0293] Construction of suitable vectors containing the desiredtherapeutic gene coding and control sequences employs standard ligationand restriction techniques, which are well understood in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

[0294] Site-specific DNA cleavage is performed by treating with thesuitable restriction etize (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by the manufacturer of these commercially availablerestriction enzymes (See, e.g. New England Biolabs Product Catalog). Ingeneral, about 1 μg of plasmid or DNA sequences is cleaved by one unitof enzyme in about 20 μl of buffer solution. Typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate.

[0295] Incubation times of about one hour to two hours at about 37° C.are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology65:499-560(1980).

[0296] Restriction cleaved fragments may be blunt ended by treating withthe large fragment of E. coli DNA polymerase I (Klenow) in the presenceof the four deoxynucleotide triphosphates (dNTPs) using incubation timesof about 15 to 25 min at 20° C. to 25° C. in 50 mM Tris (pH 7.6) 50 mMNaCl, 6 mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fillsin at 5′ sticky ends but chews back protruding 3′ single strands, eventhough the four dNTs are present. If desired, selective repair can beperformed by supplying only one of the dNTPs, or with selected dNTPs,within the limitations dictated by the nature of the sticky ends. Aftertreatment with Klenow, the mixture is extracted with phenol/chloroformand ethanol precipitated. Treatment under appropriate conditions with S1nuclease or Bal-31 results in hydrolysis of any single-stranded portion.

[0297] Ligations are performed in 10-50 μl volumes under the followingstandard conditions and temperatures using T4 DNA ligase. Ligationprotocols are standard (D. Goeddel (ed.) Gene Expression Technology:Methods in Enzymology (1991)).

[0298] In vector construction employing “vector fragments”, the vectorfragment is commonly treated with bacterial alkaline phosphatase (BAP)or calf intestinal alkaline phosphatase (CIP) in order to remove the 5′phosphate and prevent religation of the vector. Alternatively,religation can be prevented in vectors which have been double digestedby additional restriction enzyme digestion of the unwanted fragments.

[0299] Suitable vectors include viral vector systems e.g. ADV, RV, andAAV (R. J. Kaufman “Vectors used for expression in mammalian cells” inGene Expression Technology, edited by D. V. Goeddel (1991).

[0300] Many methods for inserting functional DNA transgenes into cellsare known in the art. For example, non-vector methods include nonviralphysical transfection of DNA into cells; for example, microinjection(DePamphilis et al., BioTechnigue 6:662-680 (1988)); liposomal mediatedtransfection (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417(1987), Felgner and Holm, Focus 11:21-25 (1989) and Felgner et al.,Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods knownin the art.

[0301] Administration of Modified Vectors into Subject

[0302] One way to get DNA into a target cell is to put it inside amembrane bound sac or vesicle such as a spheroplast or liposome, or bycalcium phosphate precipitation (CaPO₄) (Graham F. and Van der Eb, A.,Virology 52:456 1973; Schaefer-Ridder M., et al., Liposomes as genecarriers: Efficient transduction of mouse L cells by thymidine kinasegene. Science 1982; 215:166; Stavridis J C, et al., Construction oftransferrin-coated liposomes for in vivo transport of exogenous DNA tobone marrow erytiroblasts in rabbits. Exp Cell Res 1986; 164:568-572).

[0303] A vesicle can be constructed in such-a way that its membrane willfuse with the outer membrane of a target cell. The vector of theinvention in vesicles can home ,into the target cells. The spheroplastsare maintained in high ionic strength buffer until they can be fusedthrough the mammalian target cell using fusogens such as polyethyleneglycol.

[0304] Liposomes are artificial phospholipid vesicles. Vesicles range insize from 0.2 to 4.0 micrometers and can entrap 10% to 40% of an aqueousbuffer containing macromolecules. The liposomes protect the DNA fromnucleases and facilitate its introduction into target cells.Transfection can also occur through electroporation.

[0305] Before administration, the modified vectors are suspended incomplete PBS at a selected density for injection. In addition to PBS,any osmotically balanced solution which is physiologically compatiblewith the subject may be used to suspend and inject the modified vectorsinto the host.

[0306] For injection, the cell suspension is drawn up into the syringeand administered to anesthetized recipients. Multiple injections may bemade using this procedure. The viral suspension procedure thus permitsadministration of genetically modified vectors to any predetermined sitein the target organ (e.g., prostate), is relatively non-traumatic,allows multiple administrations simultaneously in several differentsites or the same site using the same viral suspension. Multipleinjections may consist of a mixture of therapeutic genes.

[0307] Uses of the Modified Vectors

[0308] The present invention provides methods for maintaining andincreasing expression of therapeutic genes using a fragment havingexpression activity.

[0309] The methods of the invention are exemplified by embodiments inwhich modified vectors carrying a therapeutic gene are injected into asubject.

[0310] In a first embodiment a protein product is expressed comprisinggrowing the host vector system of the invention so as to produce theprotein in the host and recovering the protein so produced. This methodpermits the expression of genes of interest in both unicellular andmulticellular organisms. For example, in an in vitro assay, prostatecells having the vector of the invention comprising a gene of interest(e.g., the ras gene) may be used in microtiter wells as an unlimited forthe ras gene product. A sample from a subject would be added to thewells to detect the presence of antibodies directed against the rasgene. This assay can aid in the quantitative and qualitativedetermination of the presence of ras antibodies in the sample for theclinical assessment of whether the subject's immune system is combattingthe disease associated with elevated levels of ras.

[0311] In a second embodiment metastatic prostate cancer is treated viagene therapy, i.e., the correction of a disease phenotype in vivothrough the use of the nucleic acid molecules of the invention.

[0312] In accordance with the practice of this invention, the subject ofthe gene therapy may be a human, equine, porcine, bovine, murine,canine, feline, or avian subject. Other mammals are also included inthis invention.

[0313] The most effective mode of administration and dosage regimen forthe molecules of the present invention depends upon the exact locationof the prostate tumor being treated, the severity and course of thecancer, the subject's health and response to treatment and the judgmentof the treating physician. Accordingly, the dosages of the moleculesshould be titrated to the individual subject. The molecules may bedelivered directly or indirectly via another cell, autologous cells arepreferred, but heterologous cells are encompassed within the scope ofthe invention.

[0314] The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m² of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided dose may beadministered daily or proportionally reduced depending on the specifictherapeutic situation).

[0315] It would be clear that the dose of the molecules of the inventionrequired to achieve treatment may be further modified with scheduleoptimization.

[0316] Generation of Transgenic Animals

[0317] Another aspect of the invention provides transgenic non-humanmammals comprising PSCA nucleic acids. For example, in one application,PSCA-deficient non-human animals can be generated using standardknock-out procedures to inactivate a PSCA homologue or, if such animalsare non-viable, inducible PSCA homologue antisense molecules can be usedto regulate PSCA homologue activity/expression. Alternatively, an animalcan be altered so as to contain a human PSCA-encoding nucleic acidmolecule or an antisense-PSCA expression unit that directs theexpression of PSCA protein or the antisense molecule in a tissuespecific fashion. In such uses, a non-human mammal, for example a mouseor a rat, is generated in which the expression of the PSCA homologuegene is altered by inactivation or activation and/or replaced by a humanPSCA gene. This can be accomplished using a variety of art-knownprocedures such as targeted recombination. Once generated, the PSCAhomologue deficient animal, the animal that expresses PSCA (human orhomologue) in a tissue specific manner, or an animal that expresses anantisense molecule can be used to (1) identify biological andpathological processes mediated by the PSCA protein, (2) identifyproteins and other genes that interact with the PSCA proteins, (3)identify agents that can be exogenously supplied to overcome a PSCAprotein deficiency and (4) serve as an appropriate screen foridentifying mutations within the PSCA gene that increase or decreaseactivity.

[0318] For example, it is possible to generate transgenic miceexpressing the human minigene encoding PSCA in a tissue specific-fashionand test the effect of over-expression of the protein in tissues andcells that normally do not contain the PSCA protein. This strategy hasbeen successfully used for other genes, namely bcl-2 (Veis et al. Cell1993 75:229). Such an approach can readily be applied to the PSCAprotein/gene and can be used to address the issue of a potentialbeneficial or detrimental effect of the PSCA proteins in a specifictissue.

[0319] Further, in another embodiment, the invention provides atransgenic animal having germ and somatic cells comprising an oncogenewhich is linked to a PSCA upstream region effective for the expressionof said gene in the tissues of said mouse for the promotion of a cancerassociated with the oncogene, thereby producing a mouse model of thatcancer.

[0320] Compositions

[0321] The invention provides a pharmaceutical composition comprising aPSCA nucleic acid molecule of the invention or an expression vectorencoding a PSCA protein or encoding a fragment thereof and, optionally,a suitable carrier. The invention additionally provides a pharmaceuticalcomposition comprising an antibody or fragment thereof which recognizesand binds a PSCA protein. In one embodiment, the antibody or fragmentthereof is conjugated or linked to a therapeutic drug or a cytotoxicagent.

[0322] Suitable carriers for pharmaceutical compositions include anymaterial which when combined with the nucleic acid or other molecule ofthe invention retains the molecule's activity and is non-reactive withthe subject's immune systems. Examples include, but are not limited to,any of the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods. Such compositions may also beformulated within various lipid compositions, such as, for example,liposomes as well as in various polymeric compositions, such as polymermicrospheres.

[0323] The invention also provides a diagnostic composition comprising aPSCA nucleic acid molecule, a probe that specifically hybridizes to anucleic acid molecule of the invention or to any part thereof, or a PSCAantibody or fragment thereof. The nucleic acid molecule, the probe orthe antibody or fragment thereof can be labeled with a detectablemarker. Examples of a detectable marker include, but are not limited to,a radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Further, theinvention provides a diagnostic composition comprising a PSCA-specificprimer pair capable of amplifying PSCA-encoding sequences usingpolymerase chain reaction methodologies, such as RT-PCR.

EXAMPLES Example 1 Identification And Molecular Characterization Of ANovel Prostate Cell Surface Antigen (PSCA)

[0324] Materials and Methods

[0325] LAPC-4 Xenografts: LAPC-4 xenografts were generated as describedin Klein et al, 1997, Nature Med. 3: 402-408.

[0326] RDA. Northern Analysis and RT-PCR: Representational differenceanalysis of androgen dependent and independent LAPC-4 tumors wasperformed as previously described (Braun et al., 1995, Mol. Cell. Biol.15: 4623-4630). Total RNA was isolated using Ultraspec^(R) RNA isolationsystems (Biotecx, Houston, Tex.) according to the manufacturer'sinstructions. Northern filters were probed with a 660 bp RDA fragmentcorresponding to the coding sequence and part of the 3′ untranslatedsequence of PSCA or a ˜400 bp fragment of PSA. The human multiple tissueblot was obtained from Clontech and probed as specified. For reversetranscriptase (RT)-PCR analysis, first strand cDNA was synthesized fromtotal RNA using the GeneAmp RNA PCR core kit (Perkin Elmer-Roche, N.J.).For RT-PCR of human PSCA transcripts, primers 5′-tgcttgccctgttgatggcag-and 3′-ccagagcagcaggecgagtgca-were used to amplify a ˜320 bp fragment.Thermal cycling was performed by 25-25 cycles of 95° for 30 sec, 60° for30 sec and 72° for 1 min, followed by extension at 72° for 10 min.Primers for GAPDH (Clontech) were used as controls. For mouse PSCA, theprimers used were 5′ -ttctcctgctggccacctac- and 3′-gcagctcatcccttcacaat-.

[0327] In Situ Hybridization Assay for PSCA mRNA: For mRNA in situhybridization, recombinant plasmid pCR II (1 ug, Invitrogen, San Diego,Calif.) containing the full-length PSCA gene was linearized to generatesense and antisense digoxigenin labeled riboprobes. In situhybridization was performed on an automated instrument (Ventana Gen II,Ventana Medical Systems) as previously described (Magi-Galluzzi et al.,1997, Lab. Invest. 76: 37-43). Prostate specimens were obtained from apreviously described database which has been expanded to ˜130 specimens(Magi-Galluzzi et al., supra). Slides were read and scored by twopathologists in a blinded fashion. Scores of 0-3 were assigned accordingto the percentage of positive cells (0=0%; 1=<25%; 2=25-50%; 3 =>50%)and the intensity of staining (0=0; 1=1+; 2=2+; 3=3+). The two scoreswere multiplied to give an overall score of 0-9.

[0328] RESULTS

[0329] Human PSCA cDNA: Representational Difference Analysis (RDA), aPCR-based subtractive hybridization technique, was used to compare geneexpression between hormone dependent and hormone independent variants ofa human prostate cancer xenograft (LAPC-4) and to isolate cDNAsupregulated in the androgen-independent LAPC-4 subline. Multiple geneswere cloned, sequenced, and checked for differential expression. One 660bp fragment (clone #15) was identified which was found to be highlyoverexpressed in xenograft tumors when compared with normal prostate.Comparison of the expression of this clone to that of PSA in normalprostate and xenograft tumors suggested that clone #15 was relativelycancer specific (FIG. 9).

[0330] Sequence analysis revealed that clone #15 had no exact match inthe databases, but shared 30% nucleotide homology with stem cell antigen2, a member of the Thy-1/Ly-6 superfamily ofglycosylphosphatidylinositol (GPI)-anchored cell surface antigens. Clone#15 encodes a 123 amino acid protein which is 30% identical to SCA-2(also called RIG-E) and contains a number of highly conserved cysteineresidues characteristic of the Ly-6/Thy-1 gene family (FIG. 3).Consistent with its homology to a family of GPI-anchored proteins, clone#15 contains both an amino-terminal hydrophobic signal sequence and acarboxyl-terminal stretch of hydrophobic amino acids preceded by a groupof small amino acids defining a cleavage/binding site for GPI linkage(Udenfriend and Kodukula, 1995, Ann. Rev. Biochem. 64: 563-591). It alsocontains four predicted N-glycosylation sites. Because of its stronghomology to the stem cell antigen-2, clone #15 was renamed prostate stemcell antigen (PSCA). 5′ and 3′ PCR RACE analysis was then performedusing cDNA obtained from the LAPC-4 androgen independent xenograft andthe full length cDNA nucleotide sequence (including the coding anduntranslated regions) was obtained. The nucleotide sequence of the fulllength cDNA encoding human PSCA is shown in FIG. 1A and the translatedamino acid sequence is shown in FIG. 1B and in FIG. 3.

[0331] PSCA is expressed in prostate cells: The distribution of PSCAmRNA in normal human tissues was examined by Northern blot analysis. Theresults, shown in FIG. 9B, demonstrate that PSCA is expressedpredominantly in prostate, with a lower level of expression present inplacenta. Small amounts of mRNA can be detected in kidney and smallintestine after prolonged exposure and at approximately 1/100th of thelevel seen in prostate tissue. RT-PCR analysis of PSCA expression innormal human tissues also demonstrates that PSCA expression isrestricted. In a panel of normal tissues, high level PSCA mRNAexpression was detected in prostate, with significant expressiondetected in placenta and tonsils (FIG. 7A). RT-PCR analysis of PSCA mRNAexpression in a variety of prostate cancer xenografts prostate cancercell lines and other cell lines, and normal prostate showed high levelexpression restricted to normal prostate, the LAPC-4 and LAPC-9 prostatecancer xenografts, and the ovarian cancer cell line A431 (FIG. 7B). Themajor PSCA transcript in normal prostate is ˜1kb (FIG. 9B). Mouse PSCAexpression was analyzed by RT-PCR in mouse spleen, liver, lung,prostate, kidney and testis. Like human PSCA, murine PSCA is expressedpredominantly in prostate. Expression can also be detected in kidney ata level similar to that seen for placenta in human tissues.

[0332] The expression of PSCA, PSMA and PSA in prostate cancer celllines and xenografts was compared by Northern blot analysis. The resultsshown in FIG. 10 demonstrate high level prostate cancer specificexpression of both PSCA and PSMA, whereas PSA expression is not prostatecancer specific.

[0333] PSCA is Expressed by a Subset of Basal Cells in Normal Prostate:Normal prostate contains two major epithelial cellpopulations--secretory luminal cells and subjacent basal cells. In situhybridizations were performed on multiple sections of normal prostateusing an antisense riboprobe specific for PSCA to localize itsexpression. As shown in FIG. 11, PSCA is expressed exclusively in asubset of normal basal cells. Little to no staining is seen in stroma,secretory cells or infiltrating lymphocytes. Hybridization with sensePSCA riboprobes showed no background staining. Hybridization with anantisense probe for GAPDH confirmed that the RNA in all cell types wasintact. Because basal cells represent the putative progenitor cells forthe terminally differentiated secretory cells, these results suggestthat PSCA may be a prostate-specific stem/progenitor cell marker(Bonkhoffet al., 1994, Prostate 24: 114-118). In addition, since basalcells are androgen-independent, the association of PSCA with basal cellsraises the possibility that PSCA may play a role in androgen-independentprostate cancer progression.

[0334] PSCA is Overexpressed in Prostate Cancer Cells: The initialanalysis comparing PSCA expression in normal prostate and LAPC-4xenograft tumors suggested that PSCA was overexpressed in prostatecancer. As demonstrated by the Northern blot analysis as shown in FIG.9, LAPC-4 prostate cancer tumors strongly express PSCA; however, thereis almost no detectable expression in sample of BPH. In contrast, PSAexpression is clearly detectable in normal prostate, at levels 2-3 timesthose seen in the LAPC-4 tumors. Thus, the expression of PSCA inprostate cancer appears to be the reverse of what is seen with PSA.While PSA is expressed more strongly in normal than malignant prostatetissue, PSCA is expressed more highly in prostate cancer.

[0335] To confirm the PSCA expression and its value in diagnosingprostate cancer, one hundred twenty six paraffin-embedded prostatecancer specimens were analyzed by mRNA in situ hybridization for PSCAexpression. Specimens were obtained from primary tumors removed byradical prostatectomy or transurethral resection in all cases exceptone. All specimens were probed with both a sense and antisense constructin order to control for background staining. Slides were assigned acomposite score as describe under Materials and Methods, with a score of6 to 9 indicating strong expression and a score of 4 meaning moderateexpression. 102/126 (81%) of cancers stained strongly for PSCA, whileanother 9/126 (7%) displayed moderate staining (FIGS. 11B and 11C). Highgrade prostatic intraepithelial neoplasia, the putative precursor lesionof invasive prostate cancer, stained strongly positive for PSCA in 82%(97/118) of specimens (FIG. 11B) (Yang et al., 1997, Am. J. Path. 150:693-703). Normal glands stained consistently weaker than malignantglands FIG. 11B). Nine specimens were obtained from patients treatedprior to surgery with hormone ablation therapy. Seven of nine (78%) ofthese residual presumably androgen-independent cancers overexpressedPSCA, a percentage similar to that seen in untreated cancers. Becausesuch a large percentage of specimens expressed PSCA mRNA, no statisticalcorrelation could be made between PSCA expression and pathologicalfeatures such as tumor stage and grade. These results suggest that PSCAmRNA overexpression is a common feature of androgen-dependent andindependent prostate cancer.

[0336] PSCA is Expressed in Androgen Independent Prostate Cancer CellLines: Although PSCA was initially cloned using subtractivehybridization, Northern blot analysis demonstrated strong PSCAexpression in both androgen-dependent and androgen-independent LAPC-4xenograft tumors (FIG. 9). Moreover, PSCA expression was detected in allprostate cancer xenografts, including the LAPC-4 and LAPC-9 xenografts.

[0337] PSCA expression in the androgen-independent, androgenreceptor-negative prostate cancer cell lines PC3 and DU145 was alsodetected by reverse-transcriptase polymerase chain reaction analysis.These data suggest that PSCA can be expressed in the absence offunctional androgen receptor.

Example 2 Biochemical Characterization Of PSCA

[0338] This experiment shows that PSCA is a glycosylated, GPI-anchoredcell surface protein.

[0339] Materials and Methods

[0340] Polyclonal Antibodies and Immunoprecipitations: Rabbit polyclonalantiserum was generated against the synthetic peptide -TARIRAVGLLTVISK-and affinity purified using a PSCA-glutathione S transferase fusionprotein. 293T cells were transiently transfected with pCDNA II(Invitrogen, San Diego, Calif.) expression vectors containing PSCA,CD59, E25 or vector alone by calcium phosphate precipitation.Immunoprecipitation was performed as previously described (Harlow andLane, 1988, Antibodies: A Laboratory Manual. (Cold Spring HarborPress)). Briefly, cells were labeled with 500uCi of-trans35S label (ICN,Irvine, Calif.) for six hours. Cell lysates and conditioned media wereincubated with lug of purified rabbit anti-PSCA antibody and 20 ulprotein A sepharose CL-4B (Pharmacia Biotech, Sweden) for two hours. Fordeglycosylation, immunoprecipitates were treated overnight at 37° with 1u N-glycosidase F (Boehringer Mannheim) or 0.1 u neuraminidase (Sigma,St. Louis, Mo.) for 1 hour followed by overnight in 2.5 mU O-glycosidase(Boehringer Mannheim).

[0341] Flow Cytometry: For flow cytometric analysis of PSCA cell surfaceexpression, single cell suspensions were stained with 2 ug/ml ofpurified anti-PSCA antibody and a 1:500 dilution of fluoresceinisothiocyanate (FITC) labeled anti-rabbit IgG (Jackson Laboratories,West Grove, Pa.). Data was acquired on a FACScan (Becton Dickinson) andanalyzed using LYSIS II software. Control samples were stained withsecondary antibody alone. Glycosylphosphatidyl inositol linkage wasanalyzed by digestion of 2×10⁶ cells with 0.5 units ofphosphatidylinositol-specific phospholipase C (PI-PLC, BoehringerMannheim) for 90 min at 37° C. Cells were analyzed prior to and afterdigestion by either FACS scanning or immunoblotting.

[0342] Results

[0343] PSCA is a GPI-Anchored Glycoprotein Expressed on the CellSurface: The deduced PSCA amino acid sequence predicts that PSCA isheavily glycosylated and anchored to the cell surface through a GPImechanism. In order to test these predictions, we produced an affinitypurified polyclonal antibody raised against a unique PSCA peptide (seeMaterials and Methods). This peptide contains no glycosylation sites andwas predicted, based on comparison to the three dimensional structure ofCD59 (another GPI-anchored PSCA homologue), to lie in an exposed portionof the mature protein (Kiefer et al., 1994, Biochem. 33: 4471-4482).Recognition of PSCA by the affinity-purified antibody was demonstratedby immunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. The polyclonalantibody immunoprecipitates predominantly a 24kd band fromPSCA-transfected, but not mock-transfected cells (FIG. 12A). Threesmaller bands are also present, the smallest being ˜10kd. Theimmunoprecipitate was treated with N and O specific glycosidases inorder to determine if these bands represented glycosylated forms ofPSCA. N-glycosidase F deglycosylated PSCA, whereas O-glycosidase had noeffect (FIG. 12A). Some GPI-anchored proteins are known to have bothmembrane-bound and secreted forms (Fritz and Lowe, 1996, Am. J. Physiol.270: G176-G183). FIG. 12B indicates that some PSCA is secreted in the293T-overexpressing system. The secreted form of PSCA migrates at alower molecular weight than the cell surface-associated form, perhapsreflecting the absence of the covalent GPI-linkage. This result mayreflect the high level of expression in the 293T cell line and needs tobe confirmed in prostate cancer cell lines and in vivo.

[0344] Fluorescence activated cell sorting (FACS) analysis was used tolocalize PSCA expression to the cell surface. Nonpermeabilizedmock-transfected 293T cells, PSCA-expressing 293T cells and LAPC-4 cellswere stained with affinity purified antibody or secondary antibodyalone. FIG. 12C shows cell surface expression of PSCA inPSCA-transfected 293T and LAPC-4 cells, but not in mock-transfectedcells. To confirm that this cell surface expression is mediated by acovalent GPI-linkage, cells were treated with GPI-specific phospholipaseC (PLC). Release of PSCA from the cell surface by PLC was indicated by agreater than one log reduction in fluorescence intensity. Recovery ofPSCA in post digest conditioned medium was also confirmed byimmunoblotting. The specificity of phospholipase C digestion forGPI-anchored proteins was confirmed by performing the same experiment on293T cells transfected with the GPI-linked antigen CD59 or the non-GPIlinked transmembrane protein E25a (Deleersnijder et al., 1996, J. Biol.Chem 271: 19475-19482). PLC digestion reduced cell surface expression ofCD59 to the same degree as PSCA but had no effect on E25. These resultssupport the prediction that PSCA is a glycosylated, GPI-anchored cellsurface protein.

Example 3 Isolation Of cDNA Encoding Murine PSCA Homologue

[0345] The human PSCA cDNA was used to search murine EST databases inorder to identify homologues for potential transgenic and knockoutexperiments. One EST obtained from fetal mouse and another from neonatalkidney were 70% identical to the human cDNA at both the nucleotide andamino acid levels. The homology between the mouse clones and human PSCAincluded regions of divergence between human PSCA and its GPI-anchoredhomologues, indicating that these clones likely represented the mousehomologue of PSCA. Alignment of these ESTs and 5′ extension usingRACE-PCR provided the entire coding sequence (FIG. 2).

Example 4 Isolation Of Human And Murine PSCA Genes

[0346] This experiment shows that PSCA is located at chromosome 8, bandq24.2.

[0347] Materials and Methods

[0348] Genomic Cloning: Lambda phage clones containing the human PSCAgene were obtained by screening a human genomic library (Stratagene)with a human PSCA cDNA probe (Sambrook et al., 1989, Molecular Cloning(Cold Spring Harbor)). BAC (bacterial artificial chromosome) clonescontaining the murine PSCA gene were obtained by screening a murine BAClibrary (Genome Systems, Inc., St. Louis, Mo.) with a murine PSCA cDNAprobe. A 14kb human Not I fragment and a 10kb murine Eco RI fragmentwere subcloned into pBluescript (Stratagene), sequenced, and restrictionmapped.

[0349] Chromosome Mapping by Fluorescence In Situ Hybridization:Fluorescence in situ chromosomal analysis (FISH) was performed aspreviously described using overlapping human lambda phage clones (Rowleyet al., 1990, PNAS USA 87: 9358-9362, H. Shizuya, PNAS USA, 89:8794).

[0350] Results

[0351] Structure of PSCA Gene: Human and murine genomic clones ofapproximately 14kb and 10kb, respectively, were obtained and restrictionmapped. A schematic representation of the gene structures of human andmurine PSCA and Ly-6/Thy-1 is shown in FIG. 8. Both the human and murinegenomic clones contain three exons encoding the translated and 3′untranslated regions of the PSCA gene. A fourth exon encoding a 5′untranslated region is presumed to exist based on PSCA's homology toother members of the Ly-6 and Thy-1 gene families (FIG. 8).

[0352] Human PSCA Gene Maps to Chromosome 8g24.2: Southern blot analysisof LAPC-4 genomic DNA revealed that PSCA is encoded by a single copygene. Other Ly-6 gene family members contain four exons, including afirst exon encoding a 5′ untranslated region and three additional exonsencoding the translated and 3′ untranslated regions. Genomic clones ofhuman and murine PSCA containing all but the presumed 5′ first exon wereobtained by screening lambda phage libraries. Mouse and human PSCAclones had a similar genomic organization. The human clone was used tolocalize PSCA by fluorescence in situ hybridization analysis.Cohybridization of overlapping human PSCA lambda phage clones resultedin specific labeling only of chromosome 8 (FIG. 13). Ninety sevenpercent of detected signals localized to chromosome 8q24, of which 87%were specific for chromosome 8q24.2. These results show that PSCA islocated at chromosome 8, band q24.2.

Example 5 Generation Of Monoclonal Antibodies Recognizing DifferentEpitopes Of PSCA

[0353] Materials and Methods

[0354] Generation and Production of Monoclonal Antibodies: BALB/c micewere immunized three times with a purified PSCA-glutathioneS-transferase (GST) fusion protein containing PSCA amino acids 22-99(FIG. 1B). Briefly, the PSCA coding sequence corresponding to aminoacids 18 through 98 of the human PSCA amino acid sequence wasPCR-amplified using the primer pair: 5′-GGAGAATTCATGGCACTGCCCTGCTGTGCTAC 3′-GGAGAATTCCTAATGGGCCCCGCTGGCGTT

[0355] The amplified PSCA sequence was cloned into pGEX-2T (Pharmacia),used to transform E. coli, and the fusion protein isolated.

[0356] Spleen cells were fused with HL-1 myeloma cells using standardhybridoma technique. Hybridomas that were positive for PSCA by ELISA andFACS analysis (see Results) were subcloned. Ascites fluid was producedin C.B. 17 scid/scid mice and monoclonal antibodies (mAbs) purifiedusing a protein G affinity column (Pharmacia Biotech, Piscataway, N.J.).PSCA mAb 1G8 was also produced in Cell-Pharm System 100 as recommendedby the manufacturer (Unisyn Technologies, Hopkinton, Mass.).

[0357] ELISA for Hybridoma Screening GST or PSCA-GST were immobilized onReacti-Bind maleic anhydride-activated polystyrene plates (Pierce,Rockford, Ill.). 50 ul of hybridoma media were added to each well andincubated for 1 hour at room temperature. Wells were washed 3 times with200 ul PBS containing 0.1% BSA and 0.05% Tween 20 and incubated for 1hour with 100 ul anti-mouse IgG (1:4000) labeled with alkalinephosphatase (Promega, Madison, Wis.). Plates were developed with analkaline phosphatase substrate (Bio-Rad, Hercules, Calif.).

[0358] Cell Culture: LNCaP was obtained from ATCC and stably transfectedwith a pCDNA II (Invitrogen) expression vector containing PSCA or vectoralone (Reiter, R. et al., 1998). 293T cells transiently transfected withPSCA or vector alone were prepared as described previously (Reiter, R.et al., 1998). LAPC-9 xenograft explants were propagated in PrEGM media(Clonetics, San Diego, Calif.) after digestion in 1% pronase for 18 min.at room temperature. Before FACS analysis, LAPC-9 cells were passedthough a 40 um cell strainer to obtain single cell suspensions.

[0359] Immunofluorescence: Cells were grown on glass coverslips coatedwith poly-L-lysine. Immunofluorescence assays were carried out onpermeabilized and nonpermeabilized fixed cells. For fixation, cells weretreated with 2% paraformaldehyde in PBS-CM (PBS, 100 uM CaCI₂, 1 mMMgCl₂) for 30 minutes in the dark, quenched with 50 uM NH₄Cl inPBS-CM-BSA (PBS, 100 uM CaCl₂, 1 mM MgCl₂, 0.2% BSA) for 10 minutes, andwashed twice with PBS-CM-BSA. For permeabilization, cells were treatedadditionally with PBS-CM-BSA-Saponin (0.075% saponin (Sigma) inPBS-CM-BSA) for 15 minutes at room temperature. Primary mAb at 2-5 mg/mlin PBS-CM-BSA (plus saponin in cases of permeabilization) was added for60 minutes and washed twice with PBS-CM-BSA. FITC-conjugated goatantimouse IgG antibody (1:500 diluted in PBS-CM-BSA +/− saponin;Southern Biotechnology, Birmingham, Ala.) was added for 30 minutes andwashed 3 times with PBS-CM. Slides were mounted in vectashield (VectorLaboratory, Inc., Burlingame, Calif.).

[0360] Flow Cytometry: Cells (1×10⁶) were incubated for 30 minutes at 4°C. with 100 ul mAb at 2 ug/ml in PBS containing 2% fetal bovine serum orhybridoma conditioned medium. After washing, cells were stained with a1:500 dilution of FITC-conjugated goat antimouse IgG (SouthernBiotechnology, Birmingham, Ala.). Data was acquired on a FACScan (BectonDickinson) and analyzed by using LYSIS II software.

[0361] Immunoblotting and Immunoprecipitation: Immunoprecipitation wasperformed as described (Harlow, E. and Lane, D., 1988). Briefly, cellswere labeled with 500uCi of trans35 label (ICN) for 6 hours. Celllysates were incubated with 3 ug mAb and 20 ul of protein A-SepharoseCL-4B (Pharmacia Biotech) for 2 hours. For immunoblotting, proteinextracts were prepared by lysing cells in 1×SDS Laemmli sample bufferand boiling for 5 min. Proteins were separated on 12.5% SDSpolyacrylamide gels and transferred to nitrocellulose membranes, washedand incubated with 2 ug mAb in 10 ml blocking buffer (5% nonfat milk inTBST). Blots were developed using the Amersham enhancedchemiluminescence detection system (Amersham, Arlington Heights, Ill.).

[0362] Immunohistochemistry: Normal formalin-fixed, paraffin-embeddedtissue samples were obtained from the Departments of Pathology atBeth-Israel Deaconess Medical Center-Harvard Medical School and UCLA.Primary radical prostatectomy specimens were selected from a previouslydescribed database (Magi-Galluzzi, C.et al., 1997). Bone metastases andmatched primary biopsy specimens were obtained from the UCLA Departmentof Pathology. Normal tissues were stained and scored independently attwo institutions in order to ensure reproducibility. Specimens obtainedfrom UCLA were stained using modifications of an immunoperoxidasetechnique previously described (Said, J. W. et al., 1998). Antigenretrieval was performed on paraffin sections using a commercial steamerand 0.01M citrate buffer pH 6.0. After incubation with PSCA mabs for 50min. (see below), slides were treated sequentially with rabbitanti-mouse IgG, swine anti-rabbit IgG and rabbit anti-swine IgG, allbiotin conjugated. Slides were then incubated withstrepavidin-peroxidase and antibody localization performed using thediaminobenzidene reaction. Specimens obtained fromBeth-Israel-Deaconess-Harvard Medical School were stained as previouslydescribed using an automated Ventana NexES instrument (Ventana MedicalSystems, Tucson, Ariz.) (Magi-Galluzzi, C. et al., 1997). Antigenretrieval was done by microwave for 15 min. in EDTA, pH 8.05 at 750W.mAbs purified at a concentration of ˜1 ug/ul from SCID ascites were usedat the following concentrations: 1G8=1:20; 3E6=1:30; 2H9=1:50;4A10=1:100; 3C5=1: 100. mAb 1G8 was produced in CellPharm System 100 andused at a concentration of 1:10. Positive controls included LAPC-9 andLNCaP-PSCA and negative controls were LNCaP and isotype-matchedirrelevant antibody. Primary biopsy specimens were available for threepatients with bone metastases. To approximate conditions ofdecalcification, slides from these specimens were treated for 20 min. inDecal-Stat (Lengers, N.Y.) prior to staining with PSCA mAbs.

[0363] Monoclonal antibodies (mAbs) were raised against a PSCA-GSTfusion protein lacking both the amino and carboxyl terminal signalsequences of PSCA. Positive fusions were selected by ELISA using thePSCA-GST fusion protein and GST alone. Out of 400 hybridomas screened,28 recognized the PSCA-GST fusion but not GST alone. These fusions werescreened secondarily by flow cytometry of nonpermeabilized 293T cellstransfected with PSCA and mock transfected 293T cells. Secondaryscreening by FACS was done in order to select clones capable ofrecognizing PSCA on the cell surface, hypothesizing that these mightlater become useful for in vivo targeting applications. Seven positivefusions were identified in this manner (mAbs 2A2, 3G3, 4A10, 1G8, 3E6,3C5 and 2H9), of which five (mAbs 4A10, 1G8, 3E6, 3C5 and 2H9) weresubcloned a purified.

[0364] The mAbs were tested for their ability to immunoprecipitate PSCAand/or to recognize PSCA on immunoblots. All mAbs were able toimmunoprecipitate PSCA from 293T-PSCA cells, as well as from LAPC-9prostate cancer xenograft tumors that express high levels of endogenousPSCA (FIG. 37). Likewise, all mAbs detected PSCA by immunoblotting,although mAbs 2H9 and 3E6 recognized only the ˜12 kd deglycosylated formof PSCA (FIG. 34).

[0365] The location on PSCA of the epitopes recognized by the five mAbswas determined by immunoblot analysis using three truncated PSCA-GSTfusions proteins. mAbs 4A10, 2H9 and 3C5 recognize an epitope residingwithin the amino-terminal portion of PSCA (i.e., amino acids 21-50); mAb1G8 recognizes an epitope within the middle region of PSCA (i.e., aminoacids 46-85); and mAb 3E6 reacts within the carboxyl-terminal portion ofPSCA (amino acids 85-99) (FIG. 15). All five mAbs are IgG as describedin FIG. 15. These results demonstrate that the five mAbs can detect PSCAin multiple assays and recognize at least three distinct epitopes onPSCA.

[0366] PSCA mAbs Stain the Cell Surface of Prostate Cancer Cells

[0367] The utility of mAbs for studying PSCA biology and for potentialclinical applications such as in vivo targeting applications isdependent on their ability to recognize the antigen of interest on theplasma membrane (Liu, H. et al., 1997; McLaughlin, P. et al., 1998; Wu,Y. et al., 1995; Tokuda, Y. et al., 1996). In order to determine theability of mAbs 2H9, 3E6, 1G8, 4A10 and C5 to recognize PSCAspecifically on the cell surface of prostate cancer cells, LNCaP cellstransfected with PSCA (LNCaP-PSCA) and LAPC-9 cells were examined byflow cytometry and indirect immunofluorescence. As with 293T-PSCA cells,all five mAbs were able to detect PSCA on the cell surface ofnonpermeabilized LNCaP-PSCA and/or LAPC-9 cells by flow cytometry (FIG.33). Mock-transfected LNCAP and LNCaP transfected with a neomycin-alonecontaining vector (LNCaP-neo), neither of which expresses detectablePSCA mRNA, were both negative.

[0368] Immunofluorescent analysis was performed on both permeabilizedand nonpermeabilized cells in order to ascertain whether PSCA proteinlocalizes to the cell surface (Liu, H. et al., 1997). NonpermeabilizedLNCaP-PSCA showed clear cell surface reactivity with mAbs 1G8, 3E6, 4A10and 3C5, but did not stain with mAb 2H9 (mAb 2H9 also did not detectPSCA on LNCaP-PSCA cells by FACS). LAPC-9 cells showed cell surfacereactivity with all five mAbs (FIG. 35). LNCaP-neo, as predicted, wasnegative both with and without permeabilization. Permeabilization ofLNCaP-PSCA and LAPC-9 resulted in both membrane and cytoplasmicstaining. All mAbs produced a punctate staining pattern on the cellsurface, which was most pronounced with mAbs 3E6, 3C5 and 4A10 (FIG.35). This pattern may reflect aggregation or clustering of PSCA toregions of the cell surface. These results demonstrate that all fivemAbs react with PSCA on the cell surface of intact prostate cancercells.

[0369] Immunohistochemical Staining of PSCA in Normal Prostate

[0370] PSCA mRNA localizes to a subset of basal cells in normalprostate, suggesting that PSCA may be a cell surface marker for prostatestem/progenitor cells (Reiter, R. et al., 1998). In order to test thepossibility that PSCA protein may be a marker of basal cells, PSCAexpression was examined immunohistochemically in paraffin-embeddedsections of normal prostate. mAbs 1G8 and 2H9 stained the cytoplasm ofboth basal and secretory cells , while mAb 3E6 reacted predominantlywith basal cells (FIG. 38). Atrophic glands, which express basal cellcytokeratins, stained strongly with all three mAbs (FIG. 38) (O'Malley,F. P. et al., 1990). mAbs 3C5 and 4A10 gave strong background stainingand/or nonspecific nuclear staining in paraffin sections and were notused further. These results suggest that although PSCA mRNA is detectedspecifically in basal cells, PSCA protein can be detected in bothepithelial cell layers (i.e. basal and secretory) of the prostate,although there are some differences in the staining patterns ofindividual antibodies.

[0371] Immunohistochemical Analysis of Normal Tissues

[0372] Our initial studies indicated that PSCA expression in men waslargely prostate-specific, with low levels of detectable RNA in kidneyand small intestine. PSCA mRNA was also detected in placenta. Theprostate-specificity of PSCA protein expression was tested byimmunohistochemical staining of 20 tissues using mAb 1G8 (see Table 1).Positive tissue staining with mAb 1G8 was confirmed with mAbs 2H9 and/or3E6 in order to ensure reproducibility with mAbs directed againstdistinct epitopes. Staining was also performed and scored independentlyat two institutions in order to confirm the results. As predicted by theRNA analysis, placenta was positive with all mAbs tested, withcytoplasmic staining detected in the trophoblasts (FIG. 39A). In kidneystaining was detected in the collecting ducts and distal convolutedtubules, but not in glomeruli (FIG. 39A). Transitional epithelium of thebladder and ureter, which had not been examined previously at the mRNAlevel, was positive with all mAbs tested (FIG. 39A). The only othertissue with significant immunoreactivity was colon, in which singlecells deep within the crypts stained intensely positive (FIG. 39A).Double staining with chromogranin indicated that these cells are ofneuroendocrine origin.

[0373] In order to confirm that mAb reactivity in bladder representedPSCA, Northern blot analysis.was performed on three normal bladdersamples obtained at radical cystectomy and compared with PSCA expressionin prostate, kidney and the LAPC-9 xenograft (FIG. 39B). PSCA mRNA wasdetected in bladder at levels lower than those seen in prostate,confirming the immunohistochemical result. No signal was detected in thethree kidney specimens, consistent with our previous observation thatPSCA expression in kidney is significantly lower than prostate (Reiter,R. et al., 1998). LAPC-9, a prostate cancer xenograft established from abone metastasis, expresses very high levels of PSCA mRNA compared withnormal bladder and prostate (Whang, Y. E. et al., 1998). These resultsconfirm that PSCA expression in men is largely prostate-predominant;however, there is also detectable PSCA protein expression in urothelium,renal collecting ducts and colonic neuroendocrine cells.

[0374] PSCA Protein is Expressed by a Majority of Localized ProstateCancers

[0375] In our previous study, mRNA was expressed in ˜80% of tumors andappeared to be expressed more highly in normal than malignant glands(Reiter, R. et al., 1998). In order to determine if PSCA protein can bedetected in prostate cancers and if PSCA protein levels are increased inmalignant compared with benign glands, paraffin-embedded pathologicalspecimens of primary and metastatic prostate cancers were immunostainedwith mAb 1G8 (FIGS. 21 and 28). Isolated cases were also stained withmAbs 3E6 or 2H9 in order to confirm the specificity of the staining.Twelve of 15 primary cancers stained positive (FIG. 21), including 2/2cases containing foci of high grade prostatic intraepithelial neoplasia.Staining intensity varied, with 7 cases showing equivalent staining incancer and adjacent normal glands and 5 showing significantly strongerstaining in cancer. In some cases there was strong expression in themalignant glands and undetectable staining in adjacent normal tissue(FIG. 21; patient 1). Also, there were some cases in which staining washeterogeneous, with some malignant glands staining more strongly thanothers (FIG. 21; patient 2). Overall, poorly differentiated tumorsstained more strongly than well differentiated ones, suggesting thatPSCA overexpression may correlate with increasing tumor grade (FIG. 21;patient 3). These results demonstrate that PSCA protein is expressed inprostate cancer. Consistent with our previous mRNA in situ studies, PSCAappears to be overexpressed in a significant percentage of cancers,perhaps in concert with increasing tumor grade.

[0376] This study describes the first characterization of PSCA proteinexpression using five monoclonal antibodies directed against PSCA.Because these mAbs recognize epitopes on the exterior of the cellsurface, they may have utility for prostate cancer diagnosis and therapy(Liu, H. et al., 1997). One possibility is that these mAbs could be usedto locate sites of metastatic disease, similar to the Prostascint scanwhich uses an antibody directed against PSMA (Sodee, D. B. et al.,1996). Another possibility is that they may be used to target prostatecancer cells therapeutically, either alone or conjugated to aradioisotope or other toxin. Similar approaches are currently beingevaluated using antibodies directed against extracellular epitopes onPSMA (Murphy, G. P. et al., 1998; Liu, H. et al., 1997; Liu, H. et al.,1998).

[0377] PSCA mAbs stain the cell surface in a punctate manner, suggestingthat PSCA may be localized to specific regions of the cell surface.GPI-anchored proteins are known to cluster in detergent-insolubleglycolipid-enriched microdomains (DIGS) of the cell surface (Varma, R.and Mayor, S., 1998). These microdomains, which include caveolae andsphingolipid-cholesterol rafts, are believed to play critical roles insignal transduction and molecular transport (Anderson, R. Caveolae,1993; Friedrichson, T. and Kurzchalia, T. V., 1998; Hoessli, D. C. andRobinson, P. J., 1998). Thy-1, a homologue of PSCA, has previously beenshown to transmit signals to src kinases through interaction inlipid-microdomains (Thomas, P. M. and Samuelson, L. E., 1992; Stefanova,I. et al., 1991). Preliminary subcellular fractionation experiments inour laboratory confirm the presence of PSCA in D1G8 (Xavier, R. et al.,1998).

[0378] GPI-anchored proteins have also been reported to localize toprostasomes, membrane-bound storage vesicles released by prostateepithelial cells (Ronquist, G. and Brody, I., 1985). CD59, aGPI-anchored inhibitor of complement-mediated cytolysis, is found inhigh concentrations in prostasomes of normal prostate epithelial cellsand prostatic secretions (Rooney, I. et al., 1993). PSCA protein isdetected in prostate secretory cells.

[0379] Contrary to our previous finding that PSCA mRNA localizedexclusively to basal cells, the current results suggest that PSCAprotein may be present in both basal and secretory cells. Similardifferences between mRNA and protein localization in prostate have beendescribed for PSMA and androgen receptor (Magi-Galuzzi, C. et al., 1997;Kawakami, M. and Nakayama, J., 1997). One possible explanation for thepresence of PSCA protein in secretory cells is that PSCA mRNA istranscribed in basal progenitor cells but that PSCA protein expressionpersists as basal cells differentiate into secretory cells. Anotherpossibility is that PSCA protein may be transferred from basal tosecretory cells posttranslationally.

[0380] Differences in staining intensity of basal and secretory cells bymAbs 3E6, 1G8 and 2H9 may reflect the distinct epitopes recognized bythe antibodies and/or differences in posttranslational modification ofPSCA in basal and secretory cells. Supporting this possibility is theobservation that the five mAbs do not react equally with PSCA in allassays or cell lines. mAb 2H9 recognizes PSCA on the cell surface ofLAPC-9 but not LNCaP-PSCA, suggesting that the epitope recognized bythis antibody may be altered or obscured in the latter cell type. Wehave also observed that mAb 3E6 does not stain cancers as strongly asmAbs 1G8 and 2H9 in some cases, suggesting that it may react withcertain forms of PSCA preferentially.

[0381] Although largely prostate-specific in men, PSCA is also expressedat lower levels in urothelium, colonic neuroendocrine cells, and renaltubules and collecting ducts. The staining seen in renal tubules andcollecting ducts is interesting in that these structures deriveembryologically from the ureteric bud of the mesonephric duct,suggesting a possible reason for the staining patterns seen in kidney.The absence of detectable PSCA mRNA in kidney specimens may reflecteither low levels of expression or the possibility that the samples wereobtained primarily from the renal cortex, whereas the collecting ductsare located in the renal medulla.

[0382] The primary impetus for identifying prostate-specific cellsurface genes is the desire to develop selective, nontoxic therapies.PSMA, another “prostate-restricted” protein, has also been shown to beexpressed in duodenum, colonic neuroendocrine cells and proximal renaltubules (Silver, D. A. et al., 1997). Preliminary reports of PSMAvaccine therapy have not produced significant toxicity (Tjoa, B. A. etal., 1998).

[0383] Expression of PSCA in urothelium and kidney appears to be lowerthan in normal prostate and significantly less than that seen in many ofthe prostate cancers evaluated. Therapies directed against PSCA maytherefore be relatively selective for cancer, much as Her-2/neuantibodies primarily target breast cancers that overexpress Her-2/neu(Disis, M. L. and Cheever, M. A., 1997).

[0384] Expression of PSCA in urothelium and kidney raises thepossibility that it may be expressed in transitional and renal cellcarcinomas. Two bladder cancers examined do express PSCA, one at levelssimilar to LAPC-9, suggesting that PSCA may be overexpressed in somecases of transitional cell carcinoma. A more complete survey of bladdercancer specimens will be required to test this possibility.

[0385] The data herein supports our earlier observation that PSCA isexpressed in a majority of prostate cancers. Likewise, PSCA protein isoverexpressed in some prostate tumors when compared to adjacent normalglands, supporting its use as a target for prostate cancer therapy. Incontrast to the mRNA in situ studies, the current results suggest thatPSCA protein expression may correlate with cancer stage and/or grade.Similar differences between RNA and protein expression have been notedfor thymosin Beta-15 (Bao, L., et al., 1996). TABLE 1 PSCA expression innormal tissues. Staining Tissue Positive Prostate (epithelium) Bladder(transitional epithelium) Placenta (trophoblasts) Colon (neuroendocrinecells) Kidney (tubules and collecting duct)* Negative Kidney (glomeruli)Prostate (stroma) Bladder (smooth muscle) Testis Endometrium Smallintestine Liver Pancreas Breast Gallbladder Skeletal muscle BrainPeripheral nerve Bone marrow Thymus Spleen Lung Bronchus Heart

Example 6 PSCA expression in prostate cancer bone metastases

[0386] This experiment shows that PSCA expression is amplified in bonemetastases of prostate cancer.

[0387] Materials and Methods

[0388] Horse Serum (NHS) (GIBCO #26050-070) was diluted (1/20 dilution)in 1% Casein, PBST. The antibodies of the invention that recognize PSCAwere diluted in 1/100 NHS, PBST.

[0389] The detection system included HRP-rabbit anti-mouse Ig (DAKOP260), HRP-swine anti-rabbit Ig (DAKO P217), HRP-rabbit anti-swine Ig(DAKO P164). Each were diluted 1/100 in 1/100 NHS, PBST.

[0390] 3,3′-diaminobenzidine tetrahyrochloride (AB) (Fluka) stock wasmade by dissolving 5 gm in 135 ml of 0.05 M Tris, pH 7.4. DAB wasaliquoted into 1 ml/vial and frozen at −20° C. A working solution of DABwas made by adding 1 ml of DAB to 40 ml of DAB buffer and 40 microlitersof 50% H₂O₂.

[0391] DAB buffer was prepared by combining 1.36 gm Imidazole (Sigma#I-0125) with 100 ml D²-H₂O, then adjusting the pH to 7.5 with 5 N HCl.After the pH adjustment 20 ml of 0.5 M Tris pH 7.4 and 80 ml of D²-H₂Owere added.

[0392] A section of a tissue/tumor known and previously demonstrated tobe positive for the antibody was run with the patient slide. This slideserved as a “positive control” for that antibody. A section of thepatient's test specimen was incubated with a negative control antibodyin place of the primary antibody. This slide served as a “negativecontrol” for the test.

[0393] The staining procedure was as follows. Bone samples were appliedto slides. The slides were then baked overnight at 60° C. Slides weredeparaffinize in 4 changes of xylene for 5 minutes each and passedthrough a graded series of ethyl alcohol (100%×4, 95%×2) to tapwaterthen transferred to NBF, and fixed for 30 minutes. The fixed slides wereplaced in running tapwater for 15 minutes, transferred to 3% H₂O₂-MeOH,incubated for 10 minutes, and washed in running tapwater for 5 minutes,then rinse in deionized water.

[0394] Slides were then subjected to 0.01 M citrate buffer pH 6.0,heated at 45° C. for 25 minutes, cooled for 15 min and then washed inPBS. The slides were then rinsed in PBS and placed onto programmed DAKOAutostainer, using the following four step program. The four stepprogram is as follows. The slide is rinsed in PBS and blocked with 1/20NHS in 1% Casein in PBST for 10 minutes. Primary antibody is thenapplied and incubated for 30 minutes followed by a buffer rinse.HRP-Rabbit anti-Mouse Ig is then applied and incubated for 15 minutesfollowed by another buffer rinse. HRP-Swine anti-Rabbit Ig is appliedand incubated for 15 minutes followed by a buffer rinse. HRP-Rabbitanti-Swine Ig is applied and incubated for 15 minutes followed by abuffer rinse.

[0395] DAB is then applied to the slide and incubated for 5 minutesfollowed by a buffer rinse. A second DAB is applied and incubated for 5minutes followed by a buffer rinse.

[0396] The slides are removed from the Autostainer and placed into slideholders, rinsed in tapwater and counter stained with Harris hematoxylin(15 seconds). The slide is then washed in tapwater, dipped inacid-alcohol, washed in tapwater, dipped in sodium bicarbonate solution,and washed in tapwater. The slides are then dehydrated in graded ethylalcohols (95%×2, 100%×3) and Propar ×3 and coverslipped with Permount.

[0397] PSCA Protein is Expressed Strongly in Prostate Cancers Metastaticto Bone

[0398] Prostate cancer is unique among human tumors in its propensity tometastasize preferentially to bone and to induce osteoblastic responses.Nine sections of prostate cancer bone metastases were examinedimmunohistochemically (FIG. 28). All reacted intensely and uniformlywith mAb 1G8 (and/or 3E6). In two instances, micrometastases not readilydetectable on hematoxylin and eosin sections could be seen afterstaining with mAb 1G8 (FIG. 28; patient 5). Overall, staining in bonemetastases was stronger and more uniform than in the primary tumors. Inthree cases, biopsy specimens from the primary tumors were available forcomparison. All were weakly positive for PSCA when compared with thematched bone metastasis, suggesting that PSCA expression was increasedin bone. In one biopsy specimen, weak staining was present in only asmall focus of malignant glands, while the remaining tumor was negative(FIGS. 21 and 28; Patient 4). In two cases, the biopsy specimens wereobtained 10 and 15 years prior to the bone metastasis, indicative of along latency period between the development of the primary andmetastatic lesions. To rule out the possibility that the strong stainingin bone was caused by the decalcification process used to prepare bonesections, the three primary biopsy specimens were also treated withdecalcification buffer. Although this treatment increased backgroundstaining, it did not alter epithelial reactivity significantly,indicating that the strong signal in bone was unlikely to be caused bythe decalcification process. These results suggest that PSCA may beselected for or upregulated in prostate cancer metastases to bone.

[0399] FIGS. 21-23 show the bone samples of bone metastases of prostatecancer were positive for PSCA. Nine sections of prostate cancer bonemetastases were examined. Consistent, intense staining was seen in nineprostate cancer bone metastases and all reacted intensely and uniformlywith mAb 1G8 (and/or 3E6). In two instances, the pathologist could notreadily identify the metastasis until staining with 1G8 highlighted thelesion. Overall, staining in bone metastases appeared stronger and moreuniform than in the primary tumors.

[0400] These results suggest that PSCA may be greatly overexpressed inprostate cancer metastases to bone. This is particularly interestingsince Sca-2, a close homologue of PSCA, was recently reported tosuppress osteoclast activity in bone marrow. If PSCA had similaractivity, it might provide one explanation for the tendency of prostatecancer metastases to produce an osteoblastic response, since inhibitionof osteoclast activity would tilt the balance of activity in bone tobone formation. Another possibility is that PSCA might be involved inadhesion to bone, since other Ly-6/Thy-1 family members are involved insimilar processes. There was heterogeneous expression of PSCA in anumber of primary prostate cancers. These results further support theuse of PSCA as a novel target for advanced disease.

[0401] One of the most intriguing results of the present study was theconsistent, intense staining seen in the nine prostate cancer bonemetastases. LAPC-9, a xenograft established from a bony metastasis, alsostained intensely for PSCA. In three patients, matched primary biopsyspecimens showed low levels of PSCA expression compared to the bonymetastases. Areas of strong PSCA expression in the primary tumors of thethree patients examined may have been missed since only biopsies wereavailable for analysis. Heterogeneous expression of PSCA was detected inat least one matched primary tumor, as well as in number of primarytumors for whom matched metastatic lesions were not available. Also, intwo cases the primary tumor was sampled at least a decade prior to thebone metastasis, raising the possibility that clones expressing highlevels of PSCA within the primary could have developed subsequent to theinitial biopsy. These results clearly demonstrate PSCA expression inbone metastases, further supporting it as a novel target for advanceddisease.

Example 7 PSCA overexpression in bladder and pancreatic carcinomas

[0402] This experiment shows that PSCA expression is higher in bladdercarcinomas than normal bladder.

[0403] Tissues from prostate, bladder, kidney, testes, and smallintestine (including prostate cancer and bladder and kidney carcinomas)were obtained from patients. -These tissues were then examined forbinding to PSCA using northern and western blot analyses as follows.

[0404] For northern blot analyses, tissue samples were excised and aless than 0.5×0.5 cm tissue sample was quick frozen in liquid nitrogen.The samples were homogenized in 7 mls of Ultraspec (Biotecx, Houston,Tex.), using a polytron homogenizer using the protocol provided byBiotecx (Ultraspec™ RNA Isolation System, Biotecx Bulletin No: 27,1992).

[0405] After quantification, 20 μg of purified RNA from each sample wereloaded onto a 1% agarose formaldehyde gel. Running and blottingconditions were the same as was used in Example 1. The filters wereseparately probed with labeled PSCA and an internal control, actin.Filters were washed and exposed for several hours-overnight.

[0406] For western blot analyses, tissue samples were excised and a lessthan 0.5×0.5 cm tissue sample was taken and quickly minced and vortexedin equal volume of hot 2×Sample Buffer (5%SDS, 20% glycerol). Sampleswere incubated at 100° for 5 mins, vortexed and clarified for 30 min.Protein concentrations were determined by Biorad's DC Protein Assay kit(Richmond, Calif.). 40 μl/sample was loaded on a 12% polyacrylamideprotein gel. Transfer to a nitrocellulose filter was done by standardmethods (Towbin et al. PNAS 76:4350 (1979). A western blot was performedby incubating the filter with IG8 primary antibody followed by asecondary antibody, i.e., a goat a mouse IgG HRP. Detection was byAmersham ECL Detection kit (Arlington Heights, Ill.).

[0407] 1G8 recognized and bound the PSCA on the cells surface of LAPC9and a bladder carcinoma (designated bladder (Rob)) in a western blotanalysis (FIG. 6). In FIG. 6, all tissues except LAPC9 were normal. Anorthern blot analysis confirmed elevated PSCA in the bladder carcinomatissue (designated bladder (Rob) (also referred to as Rob's Kid CA) andLAPC9 ) (FIG. 25).

[0408] A Northern blot analysis was performed, testing transcriptsisolated from pancreatic cancer cell lines: PANC-1 (epithelioid, ATCCNo. CRL-1469), BxPC-3 (adenocarcinoma, ATCC No. CRL-1687), HPAC(epithelial adenocarcinoma, ATCC No. CRL-2119), and Capan-1(adenocarcinoma, liver metastasis, ATCC No. HTB-79). The Northern blotwas probed with a full length cDNA clone of PSCA which detected PSCAtranscripts in two pancreatic cancer cell lines, HPAC and Capan-1 (FIG.63).

[0409] A Western blot analysis using the PSCA mAb 1G8 detected highlevels of PSCA protein in the HPAC cell line and lower levels in Capan-1and ASPC-1 (adenocarcinoma, ascites, ATCC No. CRL-1682) (FIG. 64).

Example 8 PSCA gene amplification in prostate cancer

[0410] This experiment shows that PSCA gene copy number is increasedsimilar to an increase in copy number of c-myc (FIG. 17). This isimportant because c-myc amplification correlates with poor outcome.Thus, the data suggests that PSCA amplification may also be a predictorfor poor outcome.

[0411] FISH with Chromosome Enumeration Probes and a Probe for c-Myc.

[0412] The method of FISH is well known (Qian, J. et al., “ChromosomalAnomalies in Prostatic Intraepithelial Neoplasia and Carcinoma Detectedby Fluorescence in vivo Hybridization,” Cancer Research, 1995.55:5408-5414.) Briefly, tissue sections (samples 34 and 75 were from twopatients) were deparaffinized, dehydrated, incubated in 2×SSC at 75° C.for 15 min, digested in pepsin solution [4 mg/ml in 0.9% NaCl (pH 1.5)]for 15 min at 37° C., rinsed in 2×SSC at room temperature for 5 min, andair-dried.

[0413] Directly labeled fluorescent DNA probes for PSCA and for the 8q24(c-myc) region were chosen. The PSCA cDNA (FIG. 1) was used to identifya 130 kb bacterial artificial chromosome (bac) clone (PSCA probe) thatin turn was used in the FISH analysis in accordance with themanufacturer's protocol (Genome Systems Inc.) The bac clone soidentified and used in the FISH analysis was BACH-265B12 (GenomeSystems, Inc. control number 17424).

[0414] Dual-probe hybridization was performed on the serial 5-μmsections using a SG-labeled PSCA probe together with a SO-labeled probefor 8q24 (c-myc). Probes and target DNA were denatured simultaneously inan 80° C. oven for 5 min. and each slide was incubated at 37° C.overnight.

[0415] Posthybridization washes were performed in 1.5 M urea/0.1×SSC at45° C. for 30 min and in 2×SSC at room temperature for 2 min. Nucleiwere counter-stained with 4.6-diamidino-2-phenylindole and anilfadecompound p-phenylenediamine.

[0416] The number of FISH signals was counted with a Zeiss Axioplanmicroscope equipped with a triple-pass filter (I02-104-1010; VYSIS). Thenumber of c-myc signals and PSCA signals were counted for each nucleus,and an overall mean c-myc:PSCA ratio was calculated. Results are shownin FIG. 17.

[0417] The results show that PSCA gene copy number increased in prostatecancer samples (FIG. 17). The PSCA gene is located at 8q24.2. Theincrease in gene copy number is due to both a gain in chromosome 8, andamplification of the PSCA gene (FIG. 17). Interestingly, the increase inPSCA gene copy number is similar to an increase in gene copy number ofc-myc (FIG. 17) which is also located at 8q24. A previous study hasdemonstrated that a gain of chromosome 8 and amplification of c-myc arepotential markers of prostate carcinoma progression (R B Jenkins et al1997 Cancer Research 57: 524-531).

Example 9 Reporter gene construct using the HPSCA 9 kb upstream regionto drive luciferase expression

[0418] The 14 kb Not I genomic fragment encoding the human PSCA gene wasisolated from λFIXII library encoding human genomic DNA (Stratagene), byscreening the library with a full length human PSCA cDNA probe, asdescribed in example 4 (Sambrook et al., 1989, Molecular Cloning (ColdSpring Harbor). The 14 kb human PSCA genomic fragment includes 9 kb ofPSCA upstream sequences that was used to drive expression of a reportergene.

[0419] The reporter gene vectors are depicted in FIG. 42 and wereconstructed as follows. The 14 kb Not I fragment was sub-cloned from theλ vector into a Bluescript KS vector (Stratagene), resulting in thepBSKS-PSCA (14kb) construct. The PSCA upstream sequence was subclonedfrom pBSKS-PSCA (14 kb) by PCR amplification using a primercorresponding to the T7 sequence contained within the Bluescript vector,and a primer corresponding to a sequence contained within PSCA exon 1(primer H3hPSCA3′-5, the sequence of this primer is as follows: Thesequence of H3hPSCA3′-5 is 5′-gggaagcttgcacagccttcagggtc-3′. The primercorresponding to PSCA exon 1 contained an introduced HindIII sequence toallow further subcloning following PCR amplification. The resultingamplified fragment was digested with HindIII and was subcloned into thepGL3-basic vector (Promega) to generate pGL3-PSCA (7 kb) which was usedto generate a series of deletion reporter gene constructs containingvarying lengths of PSCA upstream sequences operatively linked to theluciferase gene (FIG. 42). The deleted portions of the PSCA upstreamregions were obtained by subcloning restriction fragments from pGL3-PSCA(7 kb). The PSCA upstream region between −9 kb and −7 kb was subclonedfrom the pBSKS-PSCA (14 kb) construct, the Not I site was converted intoa blunt end by Klenow and the fragment was cloned into the SacI/HindIIIsites of pGL-PSCA (7 kb) in order to obtain the pGL3-PSCA (9 kb)construct. The reference to the sequences upstream of the PSCA codingregion, such as −9 kb and 6 kb (etc.), are relative to the ATG starttranslation codon. The reporter gene constructs pGL3-PSCA (9 kb),pGL3-PSCA (6 kb), pGL3-PSCA (3 kb), and pGL3-PSCA (1 kb) wereoperatively linked to the luciferase gene (FIG. 42). Plasmid, pGL3-CMV.contains the cytomegalovirus promoter (Boshart, M. et al., 1985 Cell41:521-530) linked to the luciferase gene and was used as a positivecontrol. Also, plasmid pGL3 contains no promoter sequence and was usedas a negative control plasmid.

Example 10 Transfection Assay Using a Reporter Gene Construct Containingthe hPSCA Upstream Region.

[0420] Triplicate dishes of prostate and non-prostate cell lines weretransfected by Tfx50 (Boeringer Manheim) with the PSCA constructpGL3-PSCA (9 kb), or the positive control construct, pGL3-CMV bothdescribed in Example 9 above, and assayed for luciferase activity (FIG.43). The cells and cell lines transfected include PrEC(androgen-independent prostate basal cell), LNCaP (androgen-dependentprostate secretory cell line), LAPC4 (androgen-dependent prostate cellline), HT1376 (bladder cell line), and 293T (kidney cell line).Expression activities of the constructs are expressed as a percentage ofthe activity of the CMV promoter. Standard errors are indicated abovethe bars.

[0421] The results show that 9 kb of human PSCA upstream sequencesdrives expression of the luciferase gene in a tissue-specific mannersimilar to the mRNA expression patterns seen for native HPSCA shown inFIG. 10 (Example 1). Luciferase was readily detectable in bothandrogen-dependent and androgen-independent prostate cell lines andbladder. Luciferase was also detectable, although at a lower level, inkidney cells.

Example 11 Identification of Regulatory Elements Within the PSCAUpstream Region

[0422] Triplicate dishes of PrEC (Clonetech) or LNCaP cells weretransfected with the reporter gene constructs or the positive controlconstruct described in Example 9 above, and assayed for luciferaseactivity. The reporter gene constructs comprise various lengths of theHPSCA upstream region operatively linked to the luciferase gene: Thepositive control construct, pGL3-CMV, comprises the CMV promoteroperatively linked to the luciferase. The cells were transfected using aTfx50 transfection system (Promega). Expression of luciferase in thetransfected cells were assayed using a Dual Luciferase Reporter AssaySystem (Promega), and the level of luciferase expression was measure arelative luciferase unit (RLU).

[0423] The ability of the various lengths of the HPSCA upstream regionto drive luciferase expression are expressed as a percentage of theactivity of the positive control construct containing the CMV promoter.Standard errors are indicated.

[0424] The results shown in FIG. 44 demonstrate that 3 kb of hPSCAupstream sequences drives expression of luciferase in both PrEc andLNCAP cells, but the level of detectable luciferase is 6 times higher inthe LNCAP cells compared to the PrEC cells. This comparison was based onthe level of detectable luciferase. In contrast, 1 kb of hPCSA upstreamsequences did not drive expression of luciferase in either cell line.

Example 12 A Targeting Vector

[0425] A targeting vector was designed to delete the endogenous PSCAcoding region, by homologous recombination. FIG. 40 depicts a targetingvector for the mouse PSCA gene, and the strategy for using the targetingvector to delete the endogenous PSCA gene contained in a mouse cell. Atargeting vector comprising a 12 kb SpeI fragment containing mouse PSCAupstream sequences, a NotI/EcoRI fragment containing the PGK promoteroperatively linked to a neo^(r) gene from the pGT-N29 vector (NewEngland BioLabs), and a 3.5 kb BstXI/XhoI fragment containing mouse PSCAdownstream sequences. Constitutive expression of the neomycin resistancegene is controlled by the PGK promoter, and allows antibiotic selectionof the targeted cells that contain the targeting vector.

[0426] As understood by one skilled in the art, the targeting vectordescribed here includes but is not limited to the neo^(r) gene forselection of the cells that contain the targeting vector or can containno selectable reporter gene. The targeting vector can also be used togenerate transgenic mice, known in the art as knock-in or knock-outmice, depending on whether the targeting vector contains a reporter geneor not, respectively. The transgenic mice can be used as an animal modelto study the function of the PSCA gene in prostate development of mice.

[0427] As an example that is not intended to be limiting, the targetingvector was used to delete the wild type endogenous genomic mouse PSCAcoding sequences in embryonic stem cells (US) cells to generate cellsthat are heterozygous, containing a deleted PSCA gene. For example, theheterozygous cells generated using the targeting vector arePSCA+/neo^(r) as shown by the results in FIG. 40. The phenotype of theheterozygous cells or transgenic mice can be compared with that of wildtype PSCA cells or animals.

[0428] The targeting vector was constructed as follows. The ends of the12 kb SpeI fragment containing the PSCA upstream and part of exon 1sequences was blunt-ended and linked to the blunt-ended NotI/EcoRIfragment from pGT-N29 (BioLabs) containing the neomycin-resistance gene.The 3′ end of the neomycin-resistance gene was linked to a blunt-ended3.5 kb BstXI/XhoI fragment containing part of PSCA exon 3 and thedownstream sequences. The resulting fragment was cloned into pGT-N29 togenerate the targeting vector pGT-N29-mPSCA5′/3′.

[0429] The targeting vector was transfected into ES cells byelectroporation using the method described in the following:Teratocarcinomas and Embryonic Stem Cells; A Practical Approach. IRLPress, Oxford (1987). Neomycin-resistant cells were selected and genomicDNA was isolated from the selected cells. A genomic Southern analysiswas performed to determine the outcome of the homologous recombinationreaction. 10 μg of DNA from the homologous recombination reaction andnon-targeted ES cells were digested with EcoRI and analyzed by theSouthern blot method (Southern, EM 1975 J. Molec. Biol. 98:503). Theblot was probed with a XhoI/EcoRI fragment that contains sequences 3′ tothe PSCA coding region. The results show that the probe detects a 10 kbfragment that corresponds to the control non-targeted cells that arePSCA+/PSCA+, and a 4 kb fragment that corresponds to the targeted cellsthat are heterozygous and contain PSCA+/neo^(r).

Example 13 Transgenic Mouse Models for Prostate Cancer

[0430] The present invention contemplates a strategy to generatetransgenic mouse models for prostate cancer, using the upstream regionsof the PSCA gene to drive expression of an oncogene, to induce tumorformation in prostate basal cells. As shown in FIG. 41, the strategyinvolves administration, e.g., microinjection, of a chimeric oncogenevector, comprising the upstream region of the PSCA gene operativelylinked to a transgene that encodes a gene product that induces formationof a tumor. Other researchers have used this technique, using differentprostate and non-prostate regulatory sequences operatively linked to anoncogene. For example, C3(1) is a prostate-predominant regulatorysequence (Moroulakou et al 1994 Proc. Nat. Acad. Sci. 91: 11236-11240)and probasin is a prostate-specific regulatory sequence (Greenberg et al1995 Proc. Nat. Acad. Sci. 92: 3439-3443), and both of these regulatorysequences drive expression of a transgene in prostate secretory cells.Cryptdin2 is a small-intestine predominant regulatory sequence(Garagenian et al Proc. Nat. Acad. Sci. 95: 15382-15387) that causedexpression of an oncogene in prostate endocrine cells. In contrast, thepresent invention contemplates using the PSCA upstream region to driveexpression of an oncogene in prostate basal cells, in order to generatea transgenic mouse model for prostate cancer.

[0431] The clinical characteristics of the induced prostate tumor can beanalyzed and compared with known characteristics of tumors caused by theparticular oncogene1 used in constructing the chimeric oncogene vector.In addition, various tissues and organs of the transgenic mouse can beanalyzed by DNA, RNA and proteins analyses to ascertain the presence andexpression patterns of the chimeric oncogene vector.

Example 14 Transgenic Mice Carrying Chimeric Vectors Comprising hPSCAUpstream Sequences and a Transgene

[0432] The expression patterns of transgenes under the control of HPSCAupstream regions will be tested. Toward this end, chimeric mice carryingchimeric vectors comprising hPSCA upstream sequences and a transgenehave been generated. Chimeric vectors comprising 9 kb or 6 kb of hPSCAupstream sequences operatively linked to a transgene were constructed,and are schematically represented in FIG. 45. The transgenes includedgreen fluorescent protein cDNA (GFP, Clontech) linked to the SV40polyadenylation sequence (PSCA (9 kb)-GFP and PSCA (6 kb)-GFP), greenfluorescent protein cDNA linked to a 3′ region of the human growthhormone that contains an intron cassette that confers stability to mRNA(PSCA (9 kb)-GFP-3′hGH and PSCA (6 kb)-GFP-3′hGH) (Brinster et al 1988PNAS 85: 836-840), and the genomic fragment encoding SV40 small andlarge T antigen including an intron (PSCA (9 kb)-SV40TAG and PSCA (6kb)-SV40TAG) (Brinster et al 1984 Cell 37:367-379).

[0433] The chimeric vectors were used to generate a line of foundertransgenic mice. Linearized chimeric vectors were microinjected intofertilized mouse eggs derived from intercrosses of C57BL/6×C3H hybridmice. Founder mice that carried the chimeric vector were identified bySouthern analysis of tail DNA, using GFP cDNA or SV40 genomic DNA as aprobe. The number of founders of each transgenic mouse line is indicatedon the right panel of FIG. 45.

Example 15 hPSCA Upstream Sequences Drives Expression of Transgene inTransgenic Mice

[0434] Two independent founder mice carrying PSCA (9 kb)-GFP transgenewere bred to Balb/c mice to obtain their offspring. At age of 8 weeksand 12 weeks, male and female transgenic or non-transgenic littermateswere sacrificed. After sacrifice, all urogenital and other tissues weretested for GFP expression by observing the fixed tissues underfluorescent illumination. The results shown in FIG. 46 show greenfluorescent images of prostate, bladder and skin tissues from a nontransgenic and a transgenic mouse. One out of two founder linesexpressed GFP protein in prostate, bladder and skin (FIG. 46). Tissuesthat did not express GFP include: seminal vesicle, liver, stomach,kidney, lung, brain, testis, pancreas, heart, skeletal muscle, smallintestine, colon, placenta.

Example 16 Transcript Expression Pattern of PSCA in Human and MouseTissue

[0435] The upper panel of FIG. 47 shows a human multiple tissue Northernblot (obtained from Clonetech), probed with a full length human PSCAcDNA probe. The results demonstrate that human PSCA transcripts areabundant in prostate, and less abundant but readily detectable inplacenta, but not detectable in spleen, thymus, testis ovary, smallintestine, colon, peripheral blood leukocytes (PBL), heart, brain, lung,liver, muscle, kidney and pancreas.

[0436] The lower panel of FIG. 47 shows an ethidium bromide-stainedagarose gel of RT-PCR analysis of murine PSCA transcript expressionpatterns in various mouse tissues. The RT-PCR was prepared usingUltraspec.RNA (Biotex), and cDNA cycle kit (Invitrogen). Primerscorresponding to a region within exon 1 and exon 3 of PSCA were used toamplify a 320 bp fragment. The exon 1 primer sequence is as follows: 5′primer: 5′-TTCTCCTGCTGGCCACCTAC-3′. The exon 3 primer sequence is asfollows: 3′ primer: 5′-GCAGCTCAATCCCTTCACAAT-3′. As a control, todemonstrate the integrity of the RNA samples isolated from the variousmouse tissues, a 300 bp G,3PD fragment was amplified.

[0437] The results shown in the lower panel of FIG. 47 demonstrate thatmurine PSCA transcripts are detectable in dorsal/lateral prostate,ventral prostate, bladder, stomach (cardiac, body and pyloric), andskin. In contrast, murine PSCA transcripts are not detectable inanterior prostate, ventral prostate, seminal vesicle, urethra, testis,kidney, duodenum, small intestine, colon, salivary gland, spleen,thymus, bone marrow, skeletal muscle, heart, brain, eye, lung and liver.The G3PDH results demonstrate that the transcripts isolated from variousmouse tissue were intact.

Example 17 Immunohistochemical Evidence of High Level Overexpression ofPSCA in Bladder Cancer

[0438] The following example demonstrates that PSCA protein is highlyoverexpressed in various grades of bladder carcinoma as determined byimmunohistochemical staining of paraffin-embedded bladder and bladdercarcinoma tissue sections using PSCA mAb 1G8.

[0439] Specifically, the following four tissues were examined: (A)normal bladder, (B) non-invasive superficial papillar, (C) carcinoma insitu (a high grade pre-cancerous lesion, (D) invasive bladder cancer.

[0440] The results are shown in FIG. 62. PSCA is expressed at low levelsin the transitional epithelium of normal bladder tissue. Very high levelexpression was detected in the carcinoma in situ sample, in all celllayers. In the invasive bladder carcinoma sample, very strong stainingwas seen, again in all cells. Lower level staining was observed in thesuperficial papillar sample. These results suggest that PSCA expressionlevels may correlate with increasing grade.

[0441] In addition to the above study, preliminary results from animmunohistochemical analysis of PSCA expression in a large number ofbladder and bladder carcinoma tissue specimens indicates the following(1) normal bladder expresses low levels of PSCA in the transitionaleptihelium; similar levels of expression are seen in low grade,papillary, noninvasive lesions; (2) carcinoma in situ, a high grade,often quite aggressive precancerous lesion, is almost always (90%)intensely positive for PSCA in all cells; (3) PSCA is expressedintensely by ˜30% of invasive cancers, i.e. overexpressed when comparedto normal bladder; and (4) metastases are intensely positive for PSCA.

Example 18 PSCA Monoclonal Antibody Mediated Inhibition of ProstateTumors in Vivo

[0442] The following examples demonstrate that unconjugated PSCAmonoclonal antibodies inhibit the growth of human prostate tumorxenografts grown in SCID mice, both when administered alone or incombination.

[0443] A. Tumor inhibition using multiple unconjugated PSCA mAbs-Study 1

[0444] Materials and Methods

[0445] Anti-PSCA Monoclonal Antibodies

[0446] Murine monoclonal antibodies were raised against a GST-PSCAfusion protein comprising PSCA amino acid residues 18-98 of the PSCAamino acid sequence (FIG. 1B) and expressed in E. coli, utilizingstandard monoclonal antibody production methods. The following sevenanti-PSCA monoclonal antibodies, produced by the corresponding hybridomacell lines deposited with the American Type Culture Collection on Dec.11, 1998, were utilized in this study: Antibody Isotype ATCC No. 1G8IgG1 HB-12612 2H9 IgG1 HB-12614 2A2 IgG2a HB-12613 3C5 IgG2a HB-126163G3 IgG2a HB-12615 4A10 IgG2a HB-12617 3E6 IgG3 HB-12618

[0447] Antibodies were characterized by ELISA, Western blot, FACS andimmunoprecipitation for their capacity to bind PSCA. FIG. 49 showsepitope mapping data for the above seven anti-PSCA mAbs as determined byELISA and Western analysis, as described in the accompanying figurelegend, demonstrating that the seven antibodies recognize differentepitopes on the PSCA protein. Immunohistochemical analysis of prostatecancer tissues and cells with these antibodies is described in Examples5 and 6 infra.

[0448] Antibody Formulation

[0449] The monoclonal antibodies described above were purified fromhybridoma tissue culture supernatants by Protein-G Sepharosechromatography, dialyzed against PBS, and stored at −20° C. Proteindeterminations were performed by a Bradford assay (Bio-Rad, Hercules,Calif.).

[0450] A therapeutic antibody cocktail comprising a mixture of the sevenindividual monoclonal antibodies, as indicated in Table 2, below, wasprepared and used for the treatment of SCID mice receiving subcutaneousinjections of LAPC-9 prostate tumor xenografts. Mouse IgG, purchasedfrom ICN (Costa Mesa, Calif.) was used as non-specific control antibody.Prior to injection into mice, all antibodies were sterilized using a0.22-micron filter. TABLE 2 Anti-PSCA Antibody Cocktail AmountMonoclonal Antibody Isotype (% of total) 1G8 IgG1 2.0 mg (16.7%) 2H9IgG1 1.0 mg (8.3%) 2A2 IgG2a 2.5 mg (20.8%) 3C5 IgG2a 2.0 mg (16.7%) 3G3IgG2a 2.5 mg (20.8%) 4A10 IgG2a 1.5 mg (12.5%) 3E6 IgG3 0.5 mg (4.2%)

[0451] Introduction of Prostate Cancer Xenografts into SCID Mice

[0452] The human prostate cancer xenograft line LAPC-9, which expressesvery high levels of PSCA, was used to produce tumors in SCID mice (PCTApplication No. WO98/16628, supra; Klein et al., 1987, supra).

[0453] For injection into IcR-SCID mice (Taconic Farms, Germantown,N.Y.), a single-cell suspension of LAPC-9 was prepared as follows. AnLAPC-9 xenograft tumor of approximately 2.0 g in size was harvested froma SCID mouse, minced into very small pieces using scissors and forceps,washed once in RPMI, and digested in a 1% solution of pronase for 20minutes. After digestion, the cell suspension was washed twice in RPMI,and resuspended in 10 ml of PrEGM medium (Clonetics, Walkersville, Md.).After overnight incubation, the cells were harvested and washed once inPrEGM, then passed through a 200-micron nylon filter to remove largeclumps and debris. Cells passing through the filter were collected,centrifuged, and resuspended in PrEGM medium. Cells were then counted,and the appropriate number of cells was transferred to a new tube,centrifuged, and resuspended at 2×concentration in RPMJ. An equal volumeof ice cold Matrigel was then added to the cell suspension, and thesuspension was kept on ice prior to injection. For injection, maleIcR-SCID mice were shaved on their flanks, and each mouse received asingle subcutaneous (s.c.) injection of 1×10⁶ cells in a volume of 100μl on the right flank. Mice injected with tumor cells were treated witheither control antibodies or the anti-PSCA monoclonal antibodypreparation as described below.

[0454] Treatment Protocol

[0455] Twenty SCID mice injected with tumor cells were treated witheither control antibodies (mouse IgG) or the anti-PSCA monoclonalantibody cocktail (above) as follows. Ten mice were treated with mouseIgG control antibody and ten mice were treated with the anti-PSCAmonoclonal antibody preparation. Injections of 200 μg of the mouse IgGcontrol antibody or the anti-PSCA monoclonal antibody cocktail wereadministered intraperitoneally on days −1,+3,+7,+11,+14, and +21relative to the injection of the tumor cells. Growth of LAPC-9 tumorswas followed by caliper measurements to determine tumor volumes on days+32,+35,+39,+42,+47,+54 and +61 relative to injection of tumor cells. Inaddition, mice were periodically bled for assaying circulating PSAlevels using a commercially available PSA test (American Qualex, SanClemente, Calif.).

[0456] One of the mice in the control group (mouse #2) expired duringthe course of the study and had no detectable tumor at the time.

[0457] Results

[0458] SCID mice receiving a subcutaneous injection of the LAPC-9prostate cancer xenograft were treated with either the anti-PSCA mAbpreparation or mouse IgG control antibody, as described above. Palpabletumors first appeared in the mouse IgG control group at 4 weeks aftertumor cell injection. Tumor volume measurements were initiated on day+32.

[0459] The results, which are tabulated in Table 3, below, as well aspresented graphically in FIG. 48, show that all of the controlmAb-treated mice developed tumors (9 out of 9 surviving, mouse #1,#3-10), but that none of the anti-PSCA mAb treated mice developed anydetectable tumor growth (0 out of 10, mouse #11-20). The control-treatedanimals developed significant tumors rapidly in most instances, andthese mice experienced constant tumor growth leading to progressivelylarger tumor sizes with time. By day 54, all control-treated mice haddeveloped detectable tumors. In sharp contrast to the control-treatedgroup, none of the ten mice treated with the anti-PSCA mAb preparationdeveloped detectable tumors, even after 61 days post xenograftinjection. TABLE 3 Recorded tumor volume (mm³) measurements DAYS MOUSE#* 32 35 39 42 47 54 61 1 416* 576 578 720 810 1045 1080 2  0  0  0  0 3100 269.5 450 476  544  648  810 4  0  0  0  0   0  87.5  151.3 5 338420 800 900 1087 1265 2002 6 216 250.3 504 476  612  850.5 1050 7 252472.5 637.5 720  720  720 1306 8 336 532 560 693 1080 1365 1617 9  0160.9 225 294  478  640  900 10  0  0 195 294  341  504  769.5 11  0  0 0  0   0   0   0 12  0  0  0  0   0   0   0 13  0  0  0  0   0   0   014  0  0  0  0   0   0   0 15  0  0  0  0   0   0   0 16  0  0  0  0   0  0   0 17  0  0  0  0   0   0   0 18  0  0  0  0   0   0   0 19  0  0 0  0   0   0   0 20  0  0  0  0   0   0   0

[0460] Clinically, the control treated mice all displayed visualsymptoms of progressively poor health as tumors developed and expanded.In contrast, the mice in the anti-PSCA mAb treatment group remainedactive, vigorous, and generally healthy in appearance throughout thetreatment period, suggesting no apparent toxicity or obviousside-effects were associated with the treatment.

[0461] In addition to tumor volume, mice were bled for determination ofcirculating PSA. Circulating PSA levels correlated with increasing tumorvolumes in the control group, whereas no detectable PSA was observed inthe anti-PSCA mAb treated group throughout the experiment.

[0462] B. Tumor Inhibition Using Multiple Unconjugated PSCA mAbs—Study 2

[0463] To verify the results described in Example 18, supra, a newlyprepared anti-PSCA mAb cocktail was evaluated for growth inhibition ofLAPC-9 tumor xenografts in vivo, essentially as described above.Briefly, a new batch of each mAb was prepared and mixed togetheraccording to the proportions presented in Table 4 . All antibodies weretested for PSCA reactivity. SCD) mice received a subcutaneous injectionof LAPC-9 xenograft cells as described above. The mice were treated witheither a cocktail of anti-PSCA mAb, or control preparations of mouse IgGor purified bovine IgG. A bovine IgG control group was included in thisstudy in order to study the effect of bovine IgG co-purified with theanti-PSCA antibodies on protein G-sepharose. Two hundred micrograms ofantibody was administered to each mouse by intraperitoneal injection ondays −1,+3,+7,+11,+14, and +21 relative to the injection of the tumorcells. Tumor volume corresponding to length (L)×(W)×(H) in mm wasmonitored by caliper measurements, and serum was collected at weeklyintervals. To determine the ellipsoid volume of the tumors, whichaccurately represents tumor mass, we used the formula L×W×H×½ (Tomaykoand Reynolds, 1989). TABLE 4 Anti-PSCA antibody cocktail 2 AmountMonoclonal Antibody Isotype (% of total) 1G8 IgG1 8.0 mg (16.7%) 2H9IgG1 4.0 mg (8.3%) 2A2 IgG2a 10.0 mg (20.8%) 3C5 IgG2a 8.0 mg (16.7%)3G3* IgG2a 10.0 mg (20.8%) 4A10 IgG2a 6.0 mg (12.5%) 3E6 IgG3 2.0 mg(4.2%)

[0464] The results of this study are presented in FIG. 53 and confirmthe results generated from the study described in Example 18-A, supra.Animals in the anti-PSCA treated group experienced significantinhibition of tumor cell growth compared with both of the controlgroups: No detectable difference in tumor growth was observed in micethat received either bovine IgG or murine IgG. The tumors in the controlgroups grew at equal rates and with similar latency. In contrast, LAPC-9tumors in mice receiving the anti-PSCA antibody cocktail exhibited alonger latency, a significantly slower rate of growth and smaller sizesat the end of the experiment. The average tumor volume at the final timepoint was 1,139 mm³ for mice treated with murine IgG (day 46), 1091 mm³for mice treated with bovine IgG (day 42) and 391 mm³ for anti-PSCAtreated mice (day 46). Due to the large tumor sizes in the bovine IgGtreated group, these mice were sacrificed earlier than mice in the othergroups. In addition, tumor volume correlated with PSA levels in theserum of the treated mice. Some mice receiving anti-PSCA antibodiesshowed very small tumors or no tumor growth at all, as was previouslyobserved in the study described in Example 1, supra. No apparenttoxicity was associated with administration of any of this antibodycocktail preparation, consistent with the study described in Example18-A.

[0465] C. Tumor Inhibition in Vivo Using Single Unconjugated PSCA mAbs

[0466] Materials and Methods

[0467] Several of the monoclonal antibodies described herein werestudied for their ability to inhibit the growth of prostate tumorxenografts in their unconjugated (or, “naked”) form using the previouslydescribed tumor challenge assay (see Examples 18-A and 18-B, above).Generally, the studies were conducted as described above, with slightmodifications as described in the results sections presented below foreach of the antibodies assayed.

[0468] C1: PSCA mAb 1G8

[0469] Anti-PSCA monoclonal antibody 1G8 is an IgG1 isotype antibody.The antitumor effect of 1G8 was evaluated using the LAPC-9 xenograft andmouse IgG as a control. The results presented in FIG. 54 demonstratethat treatment of mice with the 1G8 antibody inhibited tumor growth.Specifically, the average tumor volume at the final time point for thecontrol group was 854 mm³ versus an average tumor volume of 335 mm³ forthe 1G8 antibody treated group. These results show that the 1G8monoclonal antibody can inhibit the growth of prostate tumors when usedalone. As with the studies described supra, there was no apparenttoxicity associated with the treatment of these animals with the 1G8mAb.

[0470] The effect of the 1G8 monoclonal antibody on the growth ofprostate cancers generated with PC-3 cells was also determined. PC-3cells do not express PSCA. As shown in FIG. 65, the 1G8 antibody had noeffect on the development of PC-3 xenograft tumors, in sharp contrast toits effect on PSCA-expressing LAPC-9 xenografts. These results clearlyshow that the 1G8 antibody is inhibiting tumor cell growth through thePSCA antigen.

[0471] C2: PSCA mAbs 2A2 and 2H9

[0472] Two anti-PSCA monoclonal antibodies of different isotypes wereevaluated simultaneously for prostate tumor growth inhibition in vivo.Anti-PSCA mAbs 2A2 (IgG2a isotype) and 2H9 (IgG1 isotype) were testedfor prostate tumor inhibition as described in Example 18-Cl, immediatelyabove. The results presented in FIG. 55 demonstrate striking inhibitionof tumor cell growth in the anti-PSCA mAb treated groups versus thecontrol groups. Specifically, the average tumor volume at the final timepoint was 483 mm³ for mice treated with murine IgG (day 42), 49 mm³ formice treated with the 2A2 mAb (day 42), and 72 mm³ for the mice treatedwith 2H9 mAb (day 42). More significantly, tumor incidence was 6/6 micein the mouse IgG control group, versus 2/7 for the 2A2-treated group and1/7 for the 2H9-treated group. In the 2A2 treated group, the first tumorappeared at day 25 and the second tumor at day 42. In the 2H9 treatedgroup the single tumor present appeared at day 21. In the mouse IgGcontrol group, 4/6 of the mice had developed tumors by day 21. As withthe in vivo studies described above, there was no apparent toxicityassociated with the treatment of these animals with the 2A2 or 2H9 mAbs.

[0473] PSA levels in the serum of the treated mice were significantlylower than in control mice, and correlated directly with tumor volume(FIG. 56). At week 6, the mean PSA serum level in the mouse IgG controlgroup was 35 ng/ml, 2 ng/ml in the 2A2 group, and 8 ng/ml in the 2H9group.

[0474] This study further supports the conclusion that a single “naked”anti-PSCA monoclonal antibody is sufficient for anti-tumor activity. Inaddition, these data demonstrate that mAbs recognizing different PSCAepitopes are effective, and that the anti-tumor effect is not dependentupon a single IgG isotype since both IgG1 (1G8, 2H9) and IgG2a (2A2)mAbs inhibited tumor growth.

[0475] C3: PSCA mAbs Exert Growth Inhibitory Effect Specifically ThroughPSCA

[0476] In order to demonstrate that PSCA mAbs exert tumor growthinhibition specifically through the PSCA protein, a tumor inhibitionstudy with the 1G8 mAb and PC-3 tumor xenografts was conducted. PC-3cells do not express endogenous PSCA. This study was conducted asdescribed in Section C1 of this Example, above. The results, shown inFIG. 65, show that the PSCA mAb 1G8 had no effect on the growth of PC-3tumors in mice over a 40 day period. The results are shown, forcomparison, together with a parallel study of the effect of 1G8 onLAPC-9 prostate tumor xenografts (Example C1, above).

[0477] C4: PSCA mAb 3C5 Inhibits the Growth of Established LAPC-9Prostate Tumors in Vivo

[0478] In order to determine whether PSCA mAbs could effect growth ofestablished tumors, the following study was conducted. Briefly, a cohortof SCID mice were injected with 10⁶ LAPC-9 cells SQ, essentially asdescribed in examples C1 and C2. After tumors reached a size ofapproximately 100 cubic millimeters, mice were segregated into twogroups, a control group receiving mouse IgG and a treatment groupreceiving PSCA mAb 3C5. Each mouse was injected IP with control IgG or3C5 mAb according to the following protocol: 1 mg per injection, threetimes per week for the first 2 weeks, followed by two times per week inthe third week. Tumor volume and PSA measurements were determined asabove. The results, shown in FIG. 57, indicate that the 3C5 mAb inhibitsthe growth of established LAPC-9 prostate tumors in vivo. In at leastsome of the treatment group mice, tumor regression up to 50% of theinitial, pre-treatment size of the tumor was observed.

Example 19 In Vitro Assays for Characterizing PSCA Monoclonal Antibodies

[0479]19-A: Antibody-Dependent Cell Cytotoxicity Assay

[0480] To determine if the anti-PSCA mAbs sensitize tumor cells to ADCC,the following assay is performed. First, for NK cell mediated ADCC,spleen cells from SCID mice are cultured for 5 days in vitro asdescribed by Hooijberg et al., 1995, Cancer Res. 55: 2627-2634. Theactivated cells are then co-cultured with ⁵¹Cr-labeled LAPC-9,LNCaP-PSCA, or LNCAP target cells for four hours in the presence ofeither anti-PSCA mAbs or a control mouse IgG. LNCAP serves as a negativecontrol in all assays since it does not express PSCA. If single mAbs areused, the respective mouse IgG isotype control is also used. NK activityof the activated spleen cells is determined by incubation with themurine NK-sensitive target YAC-1. In all cases, killing is determined by⁵¹Cr-release into the medium. Spontaneous release is determined afterincubation of labeled cells only, and total release by incubation oflabeled cells with 5% Triton X-100. The percent of specific cell lysisis determined by:${\% \quad {Cell}\quad {Lysis}} = \frac{{{Experimental}^{51}{Cr}\quad {release}} - {{spontaneous}^{51}{Cr}\quad {release}}}{{{Total}^{51}{Cr}\quad {release}} - {{spontaneous}^{51}{Cr}\quad {release}}}$

[0481] 19-B: Antibody-Dependent Macrophage-Mediated Cytotoxicity Assay

[0482] To determine whether the anti-PSCA mAbs sensitize tumor cells toADMMC, the following assay is performed. Peritoneal macrophages areactivated by intraperitoneal injection of SCID mice with Brewer'sthioglycollate medium as described by Larson et al., 1988, Int. J.Cancer 42: 877-882. After four days, cells are collected byintraperitoneal lavage, and the percent of activated macrophagesdetermined by Mac-1 staining. For the assay, the activated macrophagesare co-cultured with ³H-thymidine labeled LAPC-9, LNCAP-PSCA, and LNCaPtarget cells for 48 hours in the presence of either anti-PSCA mAbs orcontrol mouse IgG. At the end of the assay, supernatants are harvestedfrom the wells and killing is determined by the amount of ³H-thymidinereleased as described above for ⁵¹Cr release.

[0483] 19-C: Complement-Mediated Tumor Cell Lysis Assay

[0484] Destruction of tumor targets by complement-dependent lysis may beperformed according to the method described by Huang et al., 1995,Cancer Res. 55: 610-616. For example, LAPC-9, LNCaP-PSCA, and LNCaPcells are labeled with ⁵¹Cr and then incubated on ice for 30′ witheither anti-PSCA mAbs or a mouse IgG control. After washing to removeunbound antibody, the cells are incubated with rabbit complement at 37°C. for 2 hr, and cell lysis measured by ⁵¹Cr-release into thesupernatant. The percent cell lysis will be determined as describedabove.

[0485] 19-D: Cell Proliferation Assay

[0486] The effect of anti-PSCA mAbs on cell proliferation may bedetermined by an MTT assay. Briefly, LNCaP-PSCA or LNCaP cells arecultured for 72 hr with varying amounts of either anti-PSCA mAbs ormouse IgG as a control. At the end of the incubation period, the cellsare washed and incubated in a solution of MTT for 4 hr. Proliferation isindicated by dehydrogenase mediated conversion of the MTT solution to apurple color and measured at a wavelength of 570 nm.

[0487] 19-E: Assay for Colony Formation in Soft Agar

[0488] Colony formation in the presence of anti-PSCA mAbs may bemeasured by growth of cells in soft agar. Briefly, 1×10⁴ LNCaP-PSCA orLNCAP cells are plated in medium containing Nobel agar. A dilutionseries of anti-PSCA mAbs is then added to plates in duplicate todetermine the effect on colony growth. Mouse IgG is used as a control.Macroscopic colonies are counted after 14-21 days in culture.

Example 20 PSCA Capture ELISA

[0489] A PSCA capture ELISA was developed in order to measure PSCAlevels in serum prior to treatment with anti-PSCA mAbs and providesinformation useful in determining the therapeutic dosage regimen. Theassay may also be useful in monitoring patient response to the therapy.

[0490] A schematic representation of the assay format is shown in FIG.50B. Briefly, affinity purified anti-PSCA peptide sheep polyclonalantibody (directed against amino acids 67-81 of the PSCA protein) andanti-PSCA monoclonal antibody 1G8 are used as capture antibodies and arecoated microtiter wells. After coating, incubation with a dilutionseries of test antigen is conducted in order to generate a standardcurve. Patient serum is added to the wells and incubated at roomtemperature. After incubation, unbound antibody is washed with PBS.Anti-PSCA monoclonal antibodies 2A2, 3C5 and 4A10 (IgG2a isotype), whichrecognize different epitopes on the PSCA protein, are used as detectionantibodies, and are added to the wells, incubated, and the wells washedto remove unbound antibody. The captured reaction is then visualized bythe addition of an anti-mouse Ig2a-horseradish peroxidase-conjugatedsecondary antibody followed by development with 3,3′ 5,5′tetramethylbenzidine base substrate and OD determinations taken.

[0491] A schematic representation of the standardization and controlantigens are shown in FIG. 50A. Briefly, a GST-fusion protein encodingamino acids 18-98 of PSCA is used for generating a standard curve forquantification of unknown samples. A secreted recombinant mammalianexpressed form of PSCA is used for quality control of the ELISA assay.This protein contains an Ig leader sequence to direct secretion of therecombinant protein and MYC and 6XHMS epitope tags for affinitypurification.

[0492] Quantification of recombinant PSCA secreted from 293T cellsengineered to express and secrete PSCA is shown in FIG. 51.

Example 21 Sequence of PSCA mAb Genes

[0493] The nucleotide sequences of the genes encoding the heavy chainvariable regions of murine monoclonal antibodies 1G8, 4A10 and 2H9 weredetermined using the methods described in Coloma et al., 1992, JImmunol. Methods 153: 89-104. Primers for heavy chain variable regionsequencing of mAbs 1G8 and 4A10 were as follows:

[0494] HLEAD. 1: ggc gat atc cac cat ggR atg Sag ctg Kgt Mat Sct ctt

[0495] CH3′: agg gaa ttc aYc tcc aca cac agg RRc cag tgg ata gac

[0496] Primers for heavy chain variable region sequencing of mAb 2H9were as follows:

[0497] HLEAD.2: ggg gat atc cac cat gRa ctt cgg gYt gag ctK ggt ttt

[0498] CH3′: agg gaa ttc aYc tcc aca cac agg RRc cag tgg ata gac

[0499] Total RNA was isolated from 1G8, 2H9, and 4A10 hybridoma cellsusing the Trizol Reagent (Gibco-BRL cat#15596). First strand synthesisreactions on 5 μg of RNA were generated using the Gibco-BRL SuperscriptII reverse transcriptase reaction according to manufacturers protocolsand CH3′. Two μl of the 30 μl first strand reaction was used in the PCRto amplify the variable regions.

[0500] First strand cDNA was synthesized from hybridoma RNA using aprimer from the constant region of the heavy chain (CH3′). The variableregion was amplified using CH3′ and a primer designed to the leadersequence (HLEAD.1 and HLEAD.2). The resulting PCR product is sequencedand the complementarity determining regions (CDRs) are determined usingthe Kabat rules. The sequences are shown in FIGS. 58, 59 and 60. Anamino acid alignment of the CDRs of these three mAbs is shown in FIG.61.

Example 22 PSCA mAb Binding Affinity

[0501] The affinity of PSCA monoclonal antibody 1G8 (described above)was determined using BIAcore™ instrumentation (Uppsala, Sweden), whichuses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect.23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) tomonitor biomolecular interactions in real time. On the, basis of generalprocedures outlined by the manufacturer (Pharmacia), kinetic analysis ofthe antibody was performed using a recombinant PSCA immobilized onto thesensor surface at a low density (30 RU). Recombinant PSCA was generatedas follows. 293T cells transiently transfected or 293 cells stablyexpressing a CMV-driven expression vector encoding PSCA with aC-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen) served assource of secreted soluble PSCA protein for purification. The HIS-taggedPSCA protein was purified over a nickel column using standardtechniques. The association and dissociation rates were determined usingthe software provided by the manufacturer. The results, tabulated below(Table 5), show that 1G8 has a 1 nanomolar K_(D), indicating a strongaffinity for the PSCA antigen. TABLE 5 BIOCORE AFFINITY DETERMINATION OFPSCA mAb 1G8 mAb in solution k_(a) (M⁻¹s⁻¹) k_(d) (M⁻¹s⁻¹) K_(D) (nM)1.68 × 10⁵ 1.69 × 10⁻⁴ 1.0

[0502] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Example 23 Immunohistochemical Analysis

[0503] This example describes immunohistochemical (IHC) analysis ofvarious formalin-fixed, paraffin-embedded tissues with the sevenanti-PSCA mAbs described supra.

[0504] Materials and Methods

[0505] Each of the seven anti-PSCA monoclonal antibodies was testedagainst: (1) a cell pellet consisting of LNCAP overexpressing PSCA,LNCaP parental, and 293T cells; (2) LAPC9AD xenograft; and (3) benignprostatic hyperplasia (BPH), prostate carcinoma, and normal prostatetissues. All tissue staining was performed by QualTek MolecularLaboratories, Inc (Santa Barbara, Calif.). Tissue blocks were sectionedat 4 microns and placed onto positively charged Capillary Gap microscopeslides (Ventana Medical Systems, Inc., Tucson, Ariz.). After dewaxing inxylene, followed by hydration through alcohol series, tissue sectionswere pretreated in a steamer for 20 minutes in the presence of sodiumcitrate (10 mM, pH 6.0) in order to optimize antibody reactivity.

[0506] After cooling for 5 minutes, the slides were immunostained usingan ABC-peroxidase technique. Briefly, slides were incubated in blockingserum (normal goat) for 5 minutes, followed by 2 μg/ml anti-PSCAmonoclonal primary antibody or 2 μg/ml mouse IgG for the negativecontrol (25 minutes), biotinylated secondary antibody-goat-anti-mouseIgG (25 min) and avidin-biotin complex (ABC) conjugated to peroxidaseenzyme, Vector Labs, Burlingame, Calif. (25 minutes). Between theincubation steps, sections were rinsed in buffer. DAB—Diaminobenzidinechromogen (QualTek Molecular Labs) was used to develop thereaction—yielding a brown precipitate. Slides were subsequentlycounterstained with hematoxylin and then coverslipped. Staining wasperformed on a TechMate 1000 automated staining instrument (VentanaMedical Systems, Inc., Tucson, Ariz.) at room temperature.

[0507] Results

[0508]FIG. 52 shows the IHC results for the anti-PSCA monoclonalantibody 3C5 in the cell pellet, LAPC9AD xenograft, a BPH sample, and aprostate carcinoma tissue (left panel). The cell pellet mix containsthree cell types of which only one, the LNCaP-PSCA cells, are expectedto stain with anti-PSCA monoclonal. As expected, 3C5 stains stronglyapproximately ⅓ of the cells. This staining conveniently shows positiveand negatively staining cell types on the same slide. The LAPC9ADxenograft is very strongly stained with 3C5 antibodies. Basal andepithelia cells in the ducts of the BPH tissue stain well but the basalcells are especially prominent. Finally, the prostate carcinoma tissueshows strong staining in the neoplastic ducts. The panel on the rightrepresents the background control consisting of pre-immune mouse serum.No background staining was detectable in any of the samples evaluated.

Example 24

[0509] Inhibition of established LAPC-9 tumor growth and prolongedsurvival following anti-PSCA antibody treatment. The LAPC-9 xenograftwas generated from a bone tumor biopsy of a patient withhormone-refractory metastatic prostate cancer.

[0510] The following examples demonstrate that anti-PSCA monoclonalantibodies, 1G8 and 3C5, inhibit the growth of established orthotopic,LAPC-9 prostate tumors, in SCID mice and significantly prolonged theirsurvival. The growth of the tumors was monitored by tracking the levelof serum PSA.

[0511] Orthotopic Injections

[0512] A single cell suspension of LAPC-9 tumors was prepared accordingto the methods described in Example 18-A. The cell suspension was mixed,at a 1:1 dilution, with Matrigel. The cell suspension was kept on iceprior to the orthotopic injections. The orthotopic injections wereperformed on male IcR-SCID mice, under anesthesia, usingketamine/xylazine. The anesthetized mice were shaved in the lowerabdomen, a 5-8 mm incision was made just above the penis to expose theabdominal wall and muscles. An incision was made through the abdominalmuscles to expose the bladder and seminal vescicles, which were thendelivered through the incision to expose the dorsal prostate. The LAPC-9cell suspension was injected into each dorsal lobe using a 500 μlHamilton syringe. Each lobe was injected with 10 μl of cell suspensioncorresponding to about 5×10⁵ cells. After the injections, both incisionswere closed using a running suture and the mice were kept under a heatlamp to recover. After the injections, the serum level of PSA wasmonitored. The mice were treated with a different regimen of 1G8 or 3C5antibody, depending on the serum level of PSA. After the antibodytreatments, the mice were kept alive, to determine the PSA levels as ameasure of the tumor growth, and to determine the survival of the micein each treatment group.

[0513] Monitoring the serum levels of PSA

[0514] The level of serum PSA was used to track the tumor size. The micewere bled on an approximate weekly basis, to monitor the levels of serumPSA, which were measured using a commercially-available PSA kit(American Qualex, San Clemente, Calif.). The mice were segregated intotwo treatment groups, based on the levels of serum PSA. For example, thegroups included: low levels of PSA (e.g., 2-3 ng/ml; FIG. 66 A); andmoderate levels of PSA (e.g., 64-78 ng/ml; FIG. 66 B). The serum PSAlevels were monitored until the tumors were visible through the abdomen.

[0515] The serum PSA levels were monitored on days+9,+15,+22,+30,+37,+44, and +51, relative to the day of injection of thetumor cells (FIG. 66 A). Similarly, the serum PSA levels were monitoredon days +13,+21,+27, and +34 (FIG. 66 B), on days +9,+16, +22,+29, and+36 (FIG. 68 A), and on days +7,+14,+21, and +28 (FIG. 68 B

[0516] Treatment with 1G8

[0517] Orthotopic, tumor-bearing mice were established according to themethods described above. Two groups of animals, exhibiting (i) lowlevels of serum PSA (2-3 ng/ml), or (ii) moderate levels of serum PSA(64-78 ng/ml) were selected for treatment.

[0518] The mice having low levels of PSA (e.g., 2-3 ng/ml) were treatedwith intraperitoneal injection of 2 mg of 1G8 antibody, three times perweek for one week, followed by 1 mg of 1G8 three times per week for thenext two weeks, followed by 1 mg of 1G8 once per week for the next threeweeks (as indicated by the arrows in FIG. 66 A).

[0519] The mice having moderate levels of PSA (e.g., 64-78 ng/ml) weretreated with intraperitoneal injection of 1 mg of 1G8 antibody, threetimes per week for three consecutive weeks, followed by a singleinjection of 1 mg of 1G8 the following week (as indicated by the arrowsin FIG. 66 B).

[0520] The control mice, having low or moderate levels of PSA, weretreated with about 0.5 to 0.8 ml of phosphate buffer solution (Gibco)(FIG. 66 A and B).

[0521] Treatment with 3C5

[0522] Similar treatments were performed on orthotopic tumor-bearingmice, using the 3C5 antibody. In the first experiment, 1 mg of 3C5antibody was administered intraperitoneally three times per week forthree consecutive weeks, followed by a single injection of 1 mg of 3C5(FIG. 68 A). In the second experiment, 2 mg of 3C5 was administeredthree times per week for the first week, followed by 1 mg three timesper week for the next two weeks (FIG. 68 B). The injections wereadministered on the days indicated by the arrows in FIG. 68 A and B.

[0523] Results—Treatment with 1G8 Results in Inhibition of Tumor Growthand Increased Survival

[0524] The serum PSA levels of the tumor-bearing mice were used to trackthe growth of the tumors, since the serum PSA levels correlated wellwith the tumor size. The mice bearing LAPC-9 AD tumors, treated with theanti-PSCA monoclonal antibody, 1G8, exhibited a reduction in the rate ofincrease in serum PSA levels (FIG. 66 A and B). This result indicatesthat 1G8 inhibits growth of tumors expressing PSCA.

[0525] In FIG. 66 A, each data point represents the mean PSA level formice receiving PBS (n=6) or 1G8 (n=6). The mice were bled on the daysindicated on the X-axis for PSA determinations.

[0526] As shown in FIG. 66 B, the mice were bled on the days indicatedon the X-axis for PSA determinations. Each data point represents themean PSA level for mice receiving PBS (n=4) or 1G8 (n=5). In FIG. 67, 4mice in the PBS-treated group and 5 mice in the 1G8-treated group.

[0527] Additionally, the mice having lower serum PSA levels that weretreated with the 1G8 antibody exhibited a reduced rate of increase inthe level of serum PSA, compared to the mice having higher serum PSAlevels that were treated with the 1G8 antibody (e.g., compare the datadescribed by (▪) in FIG. 66 A and B). This result suggests that the 1G8antibody was more effective at reducing tumor growth, when there was asmaller tumor burden, under the administration protocol of this study.

[0528] The affect of the 1G8 treatment on survival of the tumor-bearingmice was also monitored. In general, the mice treated with only PBSbegan to die within 5-6 weeks post-injection, due to local tumor growthand metastasis. In contrast, the mice treated with 1G8 antibodyexhibited a prolonged life. For example, the effect on survival was moredramatic in the mice having low serum PSA levels (FIG. 67 A), where themedian survival time in the PBS-treated mice was 56.5 days (range 42-71days) and the median survival time in the 1G8-treated mice was 89 days(range 77-101). In the mice having moderate serum PSA levels (FIG. 67 B)the median survival time in PBS-treated mice was 40 days (range 32-48days) compared to a median survival time of 78.5 days (range 52-105days) in the 1G8-treated mice. This indicates an increase of mediansurvival time of 38.5 days in 1G8-treated mice.

[0529] The inhibition of tumor growth correlated with prolonged life.Collectively, these results demonstrate that 1G8 treatment inhibitedtumor growth and increased the survival time of orthotopic tumor-bearingmice. Thus, these results suggest that treatment with 1G8 increasedsurvival time, by inhibiting tumor growth.

[0530] Treatment with 1G8 effectively inhibited tumor growth on smallerorthotopic tumors. Thus, 1G8 may represent an effective therapeutictreatment for minimal residual disease, in recurrent local disease,after primary treatment and in metastatic tumor formation.

[0531] The results of mice treated with the 3C5 antibody are similar tothe data obtained from mice treated with the 1G8 antibody. The micebearing LAPC-9 AD tumors, treated with the anti-PSCA monoclonalantibody, 3C5, exhibited a decrease in serum PSA levels. This resultindicates that 3C5 inhibits growth of tumors expressing PSCA.

[0532] Two separate experiments were conducted to evaluate the effect of3C5 treatment. The mice treated with 3C5 antibody exhibited a lower rateof increase in the level of serum PSA, compared to the mice treated withphosphate buffer solution (FIG. 68 A and B). This result suggests thatthe 3C5 antibody inhibited tumor growth.

[0533] In FIG. 68 A, each data point represents the mean PSA level formice receiving PBS (n=4) or 3C5 (n=5). In FIG. 68 B, each data pointrepresents the mean PSA level for mice receiving PBS (n=6) or 3C5 (n=6).

[0534] The mice treated with 3C5 antibody exhibited prolonged life (FIG.69 A), compared to the mice treated with PBS (FIG. 69 B). In the firstexperiment, the median survival time of the PBS-treated mice was 52 days(range 45-59 days), compared to more than 92 days (range 59 to +125days) in the 3C5-treated group (FIG. 69 A) (one mouse is still alive).In the second experiment, the median survival time in PBS-treated micewas 43 days (range 29-57), compared to 57.5 days in the 3C5-treated mice(range 33-82 days) (FIG. 69 B).

[0535] The inhibition of tumor growth correlated with prolonged life.Collectively, these results demonstrate that 3C5 treatment inhibitedtumor growth and thereby increased the survival time of orthotopictumor-bearing mice. Thus, these results indicate that treatment with 3C5increased survival time, by inhibiting tumor growth.

Example 25

[0536] Inhibition of established PC3-PSCA tumor growth and prolongedsurvival following anti-PSCA antibody treatment alone or in combinationwith doxorubicin.

[0537] The following examples demonstrate that 1G8, an anti-PSCAmonoclonal antibody, inhibited the growth of established subcutaneous,PC3-PSCA prostate tumors (AI), growing in SCID mice. Additionally, 1G8,when administered alone or in combination with doxorubicin, inhibitedthe growth of prostate tumors. Furthermore, treatment with 1G8 prolongedthe survival of these mice, when administered alone or in combinationwith doxorubicin. Treatment with 1G8 and doxorubicin appears to resultin a synergistic inhibitory effect on tumor growth and survival.

[0538] PSCA-expressing PC3 cells

[0539] PC3-PSCA cells were derived by retroviral gene transfer. Briefly,PSCA cDNA was inserted into the retroviral vector pSR-a (Muller, et al.,1991 Molec. Cell. Biol. 11:1785-1792)). Amphotropic retrovirus wasgenerated by co-transfection of 293T cells with pSR-α containing PSCAand a retroviral helper plasmid containing the amphotropic envelopeprotein. PC3 cells were subsequently infected with the PSCA containingamphotropic retrovirus, and 48 hours after infection the cells wereselected by growth in medium containing the neomycin analogue G418.After 2-3 weeks of selection and expansion, a Western blot was performedto confirm that the PC3-PSCA cells express PSCA protein. Parental PC3 orPC3 cells infected with an empty vector that did not contain PSCA wereboth negative for PSCA protein expression.

[0540] Subcutaneous Injections

[0541] PC3-PSCA cells were grown in T-150 flasks in DMEM +10% FBS priorto the injections. The cells were grown to log phase, harvested, washed,counted, then mixed with Matrigel at a 1:1 dilution, and kept on ice.For injection, male IcR-SCID mice were shaved on their flanks, and eachmouse received a single subcutaneous injection of about 1×10⁶ cells in avolume of 100 μl on the right flank. The growth of PC3-PSCA tumors wasfollowed by caliper measurements of the growing tumors. The mice wereselected for treatment when the tumor reached the size of 100-200 mm³,at approximately 9 days after the subcutaneous injection. The tumor sizewas measured at days +9,+15, +23,+29,+36, and +43 post injection.

[0542] Treatment with PBS

[0543] The control mice were treated with about 0.5 to 0.8 ml ofphosphate buffer solution (Gibco) (FIG. 66 A and B).

[0544] Treatment with 1G8

[0545] The mice treated with 1G8, were administered 1 mg of 1G8, threetimes per week for three consecutive weeks.

[0546] Treatment with Doxorubicin

[0547] An LD₅₀ experiment was performed to determine the maximumtolerable dose of doxorubicin. Doxorubicin (Sigma) and was resuspendedin sterile PBS. Doxorubicin was administered by intraperitonealinjection, at the following doses: 50 μg, 25 μg, 12.5 μg, and 6.75 μg.At the highest dose, 50 μg, all the mice died within 2 weeks. At thelower dose ranges, the mice survived for more than 4 weeks. The maximaltolerable dose was 25 μg.

[0548] The mice treated with only doxorubicin, were administered 25 μg,once weekly for three consecutive weeks.

[0549] Treatment with 1G8 and doxorubicin

[0550] The mice treated with 1G8 and doxorubicin, were administered 1 mgof 1G8 three times per week for three consecutive weeks (FIG. 70;1G8=arrows), and were administered 25 μg of doxorubicin once weekly forthree consecutive weeks (FIG. 70; doxorubicin=( )).

[0551] Results—Treatment with 1G8 alone or in Combination withDoxorubicin Results in Inhibition of Tumor Growth

[0552] The mice bearing PC3-PSCA tumors, treated with anti-PSCAmonoclonal antibody, 1G8, alone or in combination with doxorubicin,exhibited a decrease in tumor growth compared to mice treated withphosphate buffer solution or doxorubicin alone (FIG. 70). These resultsindicate that 1G8 inhibits the growth of tumors expressing PSCA. Theseresults also suggest that the combination of 1G8 and doxorubicin actsynergistically to inhibit tumor growth.

[0553] Each data point represents the mean tumor volume for micereceiving PBS (n=5), doxorubicin (n=6), 1G8 (n=6), or 1G8 +doxorubicin(n=6).

[0554] The mice treated with doxorubicin exhibited a slightly lowertumor growth rate, compared to mice treated with PBS (e.g., 4% growthinhibition at day 43). In contrast, mice treated with 1G8 antibody aloneexhibited a greater reduction in tumor growth rate, compared to the micetreated with PBS (e.g., 20% growth inhibition at day 43). The micetreated with the combination of 1G8 and doxorubicin exhibited a slightlygreater reduction in tumor growth rate, compared to the mice treatedwith PBS (e.g., 36% growth inhibition at day 43).

[0555] Treatment with 1G8 alone, in this subcutaneous model usingPC3-PSCA xenografts, effectively inhibited tumor growth on establishedandrogen-independent tumors (e.g., PC3 cells are hormone refractory).Furthermore, the combination of 1G8 and doxorubicin showed augmentedtumor growth inhibitory effects. Thus, treatment with the combination of1G8 and doxorubicin represents an effective therapeutic treatment forprostate cancer patients with metastatic disease who have failed hormoneablation therapy.

Example 26 Anti-PSCA Monoclonal Antibodies Circulate and TargetPSCA-Expressing Tumors

[0556] In one study, SCID mice bearing established, subcutaneous, LAPC-9AD tumors, described in Example 18 C4 above, were treated with eithercontrol mouse IgG or 3C5 anti-PSCA mAb as described. On day 34, 6 daysafter the last antibody treatment, the mice were sacrificed and thetumors were explanted. In a different study, tumor-bearing. SCID micewere treated with control mouse IgG or 1G8. The presence of antibody inthe tumor samples from both studies was detected by either Western blotanalysis or immunohistochemistry (IHC).

[0557] Immunohistochemistry

[0558] Explanted tumors were fixed in formalin and embedded in paraffinfor immunohistochemical analysis (performed by QualTek Molecular Labs,Santa Barbara, Calif.). The paraffin blocks were sliced, the slices werefixed on slides, and the slides were probed with biotinylated goatanti-mouse IgG followed by an avidin-biotin complex a (ABC) conjugatedto peroxidase enzyme (Vector Labs, Burlingame, Calif.). DAB(diaminobeuzidine) chromogen was used to develop the reaction whichyielded a brown precipitate. Slides were subsequently counterstainedwith hematoxylin and then coverslipped. Staining was performed on aTechMate 1000 automated staining instrument (Ventana Medical Systems,Inc., Tucson, Ariz.) at room temperature (FIG. 71).

[0559] Results—Immunohistochemistry

[0560]FIG. 71 demonstrates that the 3C5 antibody localized in the LAPC-9AD tumors from the 3C5-treated mice, but not with control IgG-treatedmice. Additionally, antibody could be detected throughout the tumor.

[0561] Western Blotting

[0562] Explanted tumors from 3 mice in the IgG-treated group and the3C5-treated group, (e.g., the mice as described in Example 18 C4 above),were lysed in boiling SDS sample buffer. The cell lysates wereelectrophoresed in SDS-PAGE gels, transferred to nitrocellulose filters,probed with goat anti-mouse IgG-HRP antibodies (Southern Biotech,Birmingham, Ala.), and visualized by enhanced chemiluminescence. Themouse IgG control antibody and 3C5 were also run on the gel as controls(FIG. 72).

[0563] In a similar experiment, LAPC-9 AD subcutaneous tumor-bearingmice were treated with either control mouse IgG or the 1G8 anti-PSCAmAb. On day 30, which was 7 days after the last antibody treatment, themice were sacrificed and tumors were explanted. Western blot analysiswas performed on explanted tumors from 3 mice in each group. Theexplanted tumors were lysed in boiling SDS sample buffer, the celllysates were electrophoresed in SDS-PAGE gels, transferred tonitrocellulose, probed with goat anti-mouse IgG-HRP antibodies (SouthernBiotech, Birmingham, Ala.), and visualized by enhancedchemiluminescence. The mouse IgG control antibody and 1G8 were also runon the gel as controls (FIG. 73).

[0564] Results—Western Blotting

[0565]FIG. 72 demonstrates that IgG heavy and light chains were readilydetected in tumor lysates from the 3C5 treated mice, but not in themouse IgG control treated mice.

[0566]FIG. 73 demonstrates that IgG heavy and light chains were readilydetected in tumor lysates from the 1G8 treated mice, but not in themouse IgG control treated mice.

[0567] These results demonstrate that anti-PSCA mAbs, such as 3C5 and1G8, can circulate and target a PSCA-expressing tumor, afteradministration to tumor-bearing mice. Furthermore the antibodylocalization is specific since control mouse IgG, which does notrecognize ,PSCA, is either absent from the tumors, or present at verylow levels when compared to tumors from anti-PSCA treated mice. Theseresults suggest that anti-PSCA mAbs have the potential to circulatethrough the body and localize to primary and metastatic, PSCA-expressingtumors in cancer patients. Furthermore, conjugated anti-PSCA mAbs may becapable of effectively destroying PSCA-expressing tumors for local,locally recurring and metastatic disease.

What is claimed is:
 1. An antibody which specifically binds to PSCA onthe surface of carcinoma cells, and is internalized within the carcinomacells to which it binds.
 2. An antibody which specifically binds to PSCAon the surface of carcinoma cells, and is cytotoxic to the carcinomacells to which it binds.
 3. An antibody which specifically binds to PSCAon the surface of carcinoma cells, and is cytostatic to the carcinomacells to which it binds.
 4. An antibody which specifically binds PSCA onthe cell surface of carcinoma cells, and is internalized and kills thecarcinoma cells to which it reacts.
 5. An antibody which specificallybinds to PSCA on the surface of carcinoma cells, and is internalized andis cytostatic to the carcinoma cells to which it binds.
 6. An antibody,comprising an antigen binding site, wherein the antigen binding siterecognizes and binds the N terminal region of PSCA.
 7. An antibody,comprising an antigen binding site, wherein the antigen binding siterecognizes and binds the C terminal region of PSCA.
 8. An antibody,comprising an antigen binding site, wherein the antigen binding siterecognizes and binds the middle region of PSCA.
 9. The antibody of claim1, 2, 3, 4, 5, 6, 7 or 8 which is a monoclonal antibody.
 10. Amonoclonal anti-idiotypic antibody reactive with an idiotype on theantibody of of claim 1, 2, 3, 4, 5, 6, 7 or
 8. 11. A recombinant proteinwhich is a murine/human chimeric antibody having (a) a variable regionof the antibody of claim 1, 2, 3, 4, 5, 6, 7 or 8 and (b) a constantregion of human origin.
 12. A polypeptide that binds PSCA comprising theantigen-binding region of the antibody of claim 1, 2, 3, 4, 5, 6, 7 or8.
 13. A monoclonal antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of the antibody ofclaim 1, 2, 3, 4, 5, 6, 7 or 8 to its target antigen.
 14. A bispecificantibody with a binding specificity for two different antigens, one ofthe antigens being that with which the antibody of claim 1, 2, 3, 4, 5,6, 7, or 8 binds.
 15. An Fab, F(ab′)2 or Fv fragment of the antibody ofclaim 1, 2, 3, 4, 5, 6, 7 or
 8. 16. A single chain antibody moleculethat binds PSCA comprising an antigen binding region of the antibody ofclaim 1, 2, 3, 4, 5, 6, 7 or
 8. 17. An immunoconjugate comprising theantibody of claim 1, 2, 3, 4, 5, 6, 7 or 8 joined to a therapeuticagent.
 18. An immunoconjugate comprising the recombinant protein ofclaim 11 joined to a therapeutic agent.
 19. An immunoconjugatecomprising the polypeptide of claim 12 joined to a therapeutic agent.20. An immunoconjugate comprising the monoclonal antibody of claim 9joined to a therapeutic agent.
 21. An immunoconjugate comprising thebispecific antibody of claim 14 joined to a therapeutic agent.
 22. Animmunoconjugate comprising the single chain antibody molecule of claim16 joined to a therapeutic agent.
 23. The immunoconjugate of any one ofclaims 17-22, wherein the therapeutic agent is a cytotoxic agent. 24.The immunoconjugate of claim 23, wherein the cytotoxic agent is selectedfrom a group consisting of ricin, ricin A-chain, doxorubicin,daunorubicin, taxol, ethiduim bromide, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, dihydroxy anthracin dione,actinomycin D, diphteria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, arbrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin; crotin,calicheamicin, sapaonaria officinalis inhibitor, maytansinoids, andglucocorticoidricin.
 25. A pharmaceutical composition useful in killinghuman cells expressing the PSCA antigen on the cell surface, comprisinga pharmaceutically effective amount of the antibody of claim 1, 2, 3, 4,5, 6, 7 or 8 and a pharmaceutically acceptable carrier.
 26. Apharmaceutical composition useful in killing human cells expressing thePSCA antigen on the cell surface, comprising a pharmaceuticallyeffective amount of the immunoconjugate of any one of the claims 17-22,and a pharmaceutically acceptable carrier.
 27. A method for treating asubject suffering from a malignant disease characterized by cells havingthe PSCA antigen on the cell surface which comprises administering tothe subject an effective amount of an immunoconjugate of any one of theclaims 17-22 such that the immunoconjugate binds the PSCA antigen andkills said cells thereby treating the subject.
 28. A method forselectively killing tumor cells expressing PSCA comprising contactingsaid tumor cells with an amount of the antibody of claim 1, 2, 3, 4, 5,6, 7 or 8 for a time sufficient to kill said cells.
 29. A method forprolonging the life of a subject with a cancer associated with PSCA,comprising administering to the subject a monoclonal antibody whichbinds to PSCA in an amount effective so as to inhibit the cancer,thereby prolonging the life of the subject.
 30. The method of claim 29,wherein said antibody is conjugated to a cytotoxic agent.
 31. The methodof claim 30, wherein said cytotoxic agent is selected from the groupconsisting of ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethiduim bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, maytansinoids, andglucocorticoidricin.
 32. The method of claim 30, wherein said cytotoxicagent is a radioactive isotope.
 33. The method of claim 32, wherein saidradioactive isotope is selected from the group consisting of ²¹²Bi,¹³¹I, ¹³¹In, ⁹⁰Y and ¹⁸⁶Re.
 34. The method of claim 29, wherein saidmonoclonal antibody is not conjugated to a cytotoxic agent.
 35. Themethod of claim 29, wherein the monoclonal antibody comprises murineantigen binding region residues and human antibody residues.
 36. Themethod of claim 29, wherein the monoclonal antibody is a humanizedantibody.
 37. The method of claim 29, wherein the monoclonal antibody isa human antibody.
 38. The method of claim 29, wherein the cancer isprostate cancer.
 39. The method of claim 29, wherein the cancer ismetastatic prostate cancer.
 40. The method of claim 29, wherein thecancer is bladder cancer.
 41. The method of claim 29, wherein the canceris a metastatic bladder cancer.
 42. The method of claim 29, wherein thecancer is a pancreatic cancer.
 43. The method of claim 42, wherein thecancer is a metastatic pancreatic cancer.
 44. The method of claim 29,further comprising administering to the patient a chemotherapeutic drug.45. The method of claim 29, further comprising administering to thepatient hormone ablation therapy.
 46. The method of claim 29, furthercomprising administering to the patient hormone antagonist therapy. 47.The method of claim 29, further comprising administering radiationtherapy to the patient.
 48. A method of inhibiting the growth of cancercells expressing PSCA, comprising administering to a patient acombination of monoclonal antibodies which bind to PSCA in an amounteffective so as to inhibit growth of the cancer cells.
 49. The method ofclaim 48, wherein the combination of monoclonal antibodies comprisemonoclonal antibodies of at least two different isotypes.
 50. The methodof claim 48, wherein the combination of monoclonal antibodies comprisemonoclonal antibodies with different epitope specificities.
 51. Themethod of claim 48, wherein the combination of monoclonal antibodiescomprises monoclonal antibodies 1G8, 2A2, 2H9, 3C5, 3E6, 3G3 and 4A10produced by the hybridomas designated HB-12612, HB-12613, HB-12614,HB-12616, HB-12618, HB-12615, and HB-12617, respectively, as depositedwith the American Type Culture Collection.
 52. The method of claim 46,wherein the combination of monoclonal antibodies is selected from thegroup consisting of mAb 1G8, 2A2, 2H9, 3C5, 3E6, 3G3 and 4A10 producedby the hybridomas designated HB-12612, HB-12613, HB-12614, HB-12616,HB-12618, HB-12615, and HB-12617, respectively, as deposited with theAmerican Type Culture Collection.